Triazine compounds useful as sorbitol dehydrogenase inhibitors

ABSTRACT

This invention is directed to sorbitol dehydrogenase inhibitory compounds of formula I 
                 
 
wherein R 1 , R 2  and R 3  are as defined in the specification. This invention is also directed to pharmaceutical compositions containing these compounds and to methods of treating or preventing diabetic complications, particularly diabetic neuropathy, diabetic nephropathy, diabetic microangiopathy, diabetic macroangiopathy, diabetic cardiomyopathy and foot ulcers. This invention is also directed to pharmaceutical compositions comprising a combination of a compound of formula I of the present invention with a second pharmaceutical agent, including an aldose reductase inhibitor, a sodium hydrogen ion exchange inhibitor, a glycogen phosphorylase inhibitor, a selective serotonin reuptake inhibitor, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, an angiotensin converting enzyme inhibitor, a thiazolidinedione antidiabetic agent, an angiotensin II receptor antagonist, a γ-aminobutyric acid agonist, a phosphodiesterase type 5 inhibitor, an adenosine agonist, and a CETP inhibitor and to methods of using these compositions.

This application claims the benefit of U.S. Provisional Application No.60/280,050, filed Mar. 30, 2001.

FIELD OF THE INVENTION

The present invention relates to novel triazine compounds of formula I,isomers thereof, prodrugs of said compounds or isomers, orpharmaceutically acceptable salts of said compounds, isomers orprodrugs, and to methods of using such compounds to inhibit sorbitoldehydrogenase (SDH), lower fructose levels or treat diabeticcomplications, such as diabetic neuropathy, diabetic retinopathy,diabetic nephropathy, diabetic cardiomyopathy, diabetic microangiopathyand diabetic macroangiopathy, in mammals. The present invention alsorelates to pharmaceutical compositions containing such triazinecompounds. The present invention also relates to pharmaceuticalcompositions and kits comprising a combination of a sorbitoldehydrogenase inhibitor of formula I, an isomer thereof, a prodrug ofsaid compound or isomer, or a pharmaceutically acceptable salt of saidcompound, isomer or prodrug, and a second pharmaceutical agent and tomethods of using these combination compositions and kits.

BACKGROUND OF THE INVENTION

Triazine compounds of formula I, as defined below, and theirpharmaceutically acceptable salts, lower fructose levels in the tissuesof mammals affected by diabetes (e.g., nerve, kidney and retina tissue)and are useful in the treatment and prevention of the diabeticcomplications referred to above. These compounds, and/or theirmetabolites in vivo, are inhibitors of the enzyme sorbitoldehydrogenase, which catalyzes the oxidation of sorbitol to fructose.

Commonly assigned U.S. Pat. Nos. 5,728,704 and 5,866,578 disclosecompounds of formula A

wherein R¹ through R⁵ are defined as disclosed therein, which are usefulas sorbitol dehydrogenase inhibitors, having utility in the treatment ofdiabetic complications. Commonly assigned International Publication No.WO 00/59510 discloses aminopyrimidines as sorbitol dehydrogenaseinhibitors. Commonly assigned published European Patent Application EP 1041 068 discloses pyrimidine derivatives as sorbitol dehydrogenaseinhibitors, useful for treating or preventing diabetic complications.

Commonly assigned U.S. non-provisional Ser. No. 09/974,414, filed Oct.9, 2001, discloses pharmaceutical combinations of statins and sorbitoldehydrogenase inhibitors. Commonly assigned U.S. non-provisional Ser.No. 09/997,039, filed, Nov. 29, 2001, discloses the combination of aγ-aminobutyric acid (GABA) agonists and sorbitol dehydrogenaseinhibitors.

U.S. Pat. Nos. 5,138,058 and 5,215,990 disclose piperazine substitutedpyrimidine compounds, having utility as tools in screening for aldosereductase inhibitors due to the sorbitol accumulating activity of saidcompounds.

SUMMARY OF THE INVENTION

The present invention is directed to compounds of formula I

an isomer thereof, a prodrug of said compound or isomer, or apharmaceutically acceptable salt of said compound, isomer or prodrug;

wherein R¹ is a) hydrogen or b) —(C₁-C₄)alkyl;

R² and R³ are each independently a) hydrogen, b) —(C₁-C₄)alkyl, c)—(C₃-C₆)cycloalkyl or d) phenyl which for each occurrence is optionallysubstituted with one or two substituents, each substituent isindependently selected from Group Q;

X is a) —C(O)—R⁴-Z, b) —SO₂—R⁴-Z, c) —C(O)—NR⁵R⁶, d) —SO₂—NR⁵R⁶ or e)1,3,5-triazin-2-yl having R^(z1) and R^(z2) substituents;

R⁴ is a) a covalent bond or b) —(C₁-C₄)alkyl-;

Z is a) phenyl or benzyl wherein the phenyl ring in each of these groupsis optionally substituted with one or two substitutents, eachsubstituent is independently selected from Group Q, or b) Het;

R⁵ and R⁶ are each independently a) hydrogen, b) —(C₁-C₄)alkyl or c)(C₃-C₆)cycloalkyl; or R⁵ and R⁶ are taken together along with thenitrogen atom to which they are attached to form pyrrolidinyl orpiperidinyl;

Het is a) pyridyl, b) thiazolyl, c) oxazolyl, d) quinolyl, e)isoquinolyl, f) phthalizinyl, g) quinoxalyl, h) benzthiazolyl, i)benzoxazolyl, j) benzofuranyl, k) benzothienyl, l) furanopyridyl or m)thienopyridyl; wherein each of these groups is optionally substitutedwith one or two substituents, each substituent is independently selectedfrom Group Q;

Group Q is a) fluoro, b) chloro, c) bromo, d) —(C₁-C₄)alkyl, e)—(C₃-C₆)cycloalkyl, f) —O—(C₁-C₄)alkyl, g) —S—(C₁-C₄)alkyl, h)—SO₂—(C₁-C₄)alkyl, i) hydroxy or j) —(C₁-C₄)alkyl-hydroxy;

R^(z1) and R^(z2) are each independently selected from a) hydrogen, b)hydroxy, c) chloro, d) —(C₁-C₄)alkyl, e) —(C₃-C₆)cycloalkyl, f)—O—(C₁-C₄)alkyl, g) —(C₁-C₄)alkyl-O—(C₁-C₄)alkyl, h) —CHO, i)—C(O)—(C₁-C₄)alkyl, j) —(C₁-C₄)alkyl-hydroxy, k) phenyl which for eachoccurrence is optionally substituted with one or two substitutents, eachsubstituent is independently selected from Group Q, l) pyrroyl, m)imidazolyl or n) triazolyl.

More particularly, the present invention provides such compounds whereinR¹ is hydrogen or methyl.

More particularly, the present invention provides such compounds whereinR²and R³ are each independently a) hydrogen, b) —(C₁-C₄)alkyl, c)—(C₃-C₆)cycloalkyl; or d) phenyl optionally substituted with one or twosubstituents, each substituent is independently selected from 1)—(C₁-C₄)alkyl, 2) —(C₃-C₆)cycloalkyl, 3) —O—(C₁-C₄)alkyl, 4) fluoro or5) chloro.

More particularly, the present invention provides such compounds whereinR² and R³ are each independently hydrogen or methyl.

Even more particularly, the present invention provides such compoundswherein R² is hydrogen and R³ is hydrogen.

Even more particularly, the present invention provides such compoundswherein R² is methyl and R³ is methyl.

More particularly, the present invention provides such compounds offormula I wherein X is 1,3,5-triazin-2-yl having R^(z1) and R^(z2)substituents.

Even more particularly, the present invention provides such compoundswherein one of the R^(z1) and R^(z2) substituents is hydrogen and theother is methyl, cyclopropyl, —CH₂OH, —CH(CH₃)OH or phenyl.

Even more particularly, the present invention provides such compoundswherein one of the R^(z1) and R^(z2) substituents is methyl and theother is methoxy or phenyl optionally substituted with 2-hydroxy.

Even more particularly, the present invention provides such compoundswherein one of the R^(z1) and R^(z2) substituents is hydroxy and theother is methyl or phenyl.

More particularly, the present invention provides such compounds offormula I wherein X is —SO₂N(CH₃)₂.

More particularly, the present invention provides such compounds offormula I wherein X is —C(═O)-benzofuranyl.

More particularly, the present invention provides such compounds offormula I wherein X is —C(═O)-furanopyridyl.

The present invention also provides pharmaceutical compositionscomprising a compound of formula I, a prodrug thereof or apharmaceutically acceptable salt of said compound or said prodrug and apharmaceutically acceptable carrier or diluent.

The present invention also provides methods of inhibiting sorbitoldehydrogenase in a mammal in need of such inhibition comprisingadministering to said mammal a sorbitol dehydrogenase inhibiting amountof a compound of formula I, a prodrug thereof or a pharmaceuticallyacceptable salt of said compound or said prodrug.

The present invention also provides methods of treating diabetes in amammal suffering from diabetes comprising administering to said mammalan effective amount of a compound of formula I, a prodrug thereof or apharmaceutically acceptable salt of said compound or said prodrug.

The present invention also provides methods of treating diabeticcomplications in a mammal comprising administering to said mammal aneffective amount of a compound of formula I, a prodrug thereof or apharmaceutically acceptable salt of said compound or said prodrug. Thepresent invention also provides such methods wherein said mammal issuffering from diabetes. The present invention also provides suchmethods wherein said diabetic complication is diabetic neuropathy. Thepresent invention also provides such methods wherein said diabeticcomplication is diabetic nephropathy. The present invention alsoprovides such methods wherein said diabetic complication is diabeticretinopathy. The present invention also provides such methods whereinsaid diabetic complication is foot ulcers. The present invention alsoprovides such methods wherein said diabetic complication is acardiovascular condition.

The combination aspects of the present invention include any and/or allof the following: the composition aspect of this invention wherein acomposition comprises a first compound of formula I, a prodrug of saidfirst compound or a pharmaceutically acceptable salt of said firstcompound or said prodrug, and a second compound, a prodrug thereof or apharmaceutically acceptable salt of said second compound or saidprodrug; the kit aspects of this invention; and, the therapeutic methodaspect of this invention wherein the methods comprise administering afirst compound of formula I, a prodrug of said first compound or apharmaceutically acceptable salt of said first compound or said prodrug,and a second compound, a prodrug thereof or a pharmaceuticallyacceptable salt of said second compound or said prodrug.

Thus, the combination aspects of the present invention include, forexample, pharmaceutical compositions comprising a compound of formula I,a prodrug thereof or a pharmaceutically acceptable salt of said compoundor said prodrug, and a second compound selected from an aldose reductaseinhibitor, a sodium hydrogen ion exchange (NHE-1) inhibitor, a glycogenphosphorylase inhibitor (GPI), a selective serotonin reuptake inhibitor,a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, anangiotensin converting enzyme inhibitor, a thiazolidinedioneantidiabetic agent, an angiotensin II receptor antagonist, aγ-aminobutyric acid (GABA) agonist, a phosphodiesterase type 5inhibitor, an adenosine agonist, a CETP inhibitor, a prodrug thereof ora pharmaceutically acceptable salt of said second compound or saidprodrug. The present invention also provides such compositionsadditionally comprising a pharmaceutically acceptable carrier ordiluent.

The combination aspects of the present invention also include kitscomprising: a.) a compound of formula I, a prodrug thereof or apharmaceutically acceptable salt of said compound or said prodrug in afirst unit dosage form; b.) an aldose reductase inhibitor, a sodiumhydrogen ion exchange (NHE-1) inhibitor, a glycogen phosphorylaseinhibitor (GPI), a selective serotonin reuptake inhibitor, a3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, anangiotensin converting enzyme inhibitor, a thiazolidinedioneantidiabetic agent, an angiotensin II receptor antagonist, aγ-aminobutyric acid (GABA) agonist, a phosphodiesterase type 5inhibitor, an adenosine agonist, a CETP inhibitor, a prodrug thereof ora pharmaceutically acceptable salt of said second compound or saidprodrug, in a second unit dosage form; and c.) a container

The combination aspects of the present invention also include methods oftreating diabetic complications in a mammal comprising administering tosaid mammal an effective amount of a compound of formula I, a prodrugthereof or a pharmaceutically acceptable of said compound or saidprodrug, and a second compound selected from an aldose reductaseinhibitor, a sodium hydrogen ion exchange (NHE-1) inhibitor, a glycogenphosphorylase inhibitor (GPI), a selective serotonin reuptake inhibitor,a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, anangiotensin converting enzyme inhibitor, a thiazolidinedioneantidiabetic agent, an angiotensin II receptor antagonist, aγ-aminobutyric acid (GABA) agonist, a phosphodiesterase type 5inhibitor, an adenosine agonist, a CETP inhibitor, a prodrug thereof ora pharmaceutically acceptable salt of said second compound or saidprodrug. The present invention also provides such methods wherein saidmammal is suffering from diabetes. The present invention also providessuch methods wherein said diabetic complication is diabetic neuropathy.The present invention also provides such methods wherein said diabeticcomplication is diabetic nephropathy. The present invention alsoprovides such methods wherein said diabetic complication is diabeticretinopathy. The present invention also provides such methods whereinsaid diabetic complication is foot ulcers. The present invention alsoprovides such methods wherein said diabetic complication is acardiovascular condition.

In a preferred embodiment of the combination aspects of the presentinvention, the second compound comprises an aldose reductase inhibitor,preferably in an aldose reductase inhibiting amount.

In an additional preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a sodium hydrogen ionexchange (NHE-1) inhibitor, preferably in a NHE-1 inhibiting amount.

In an additional preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a glycogenphosphorylase inhibitor, preferably in a glycogen phosphorylaseinhibiting amount.

In a further preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a selective serotoninreuptake inhibitor, preferably in a selective serotonin reuptakeinhibiting amount.

In a further preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, preferably ina 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibiting amount.

In another preferred embodiment of the combination aspects of thepresent invention, the second compound comprises an angiotensinconverting enzyme inhibitor, preferably in an angiotensin convertingenzyme inhibiting amount.

In a further preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a thiazolidinedioneantidiabetic agent, preferably in an insulin sensitivity increasingamount.

In another preferred embodiment of the combination aspects of thepresent invention, the second compound comprises an angiotensin IIreceptor antagonist, preferably in an angiotensin II receptor blockingamount.

In a further preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a γ-aminobutyric acid(GABA) agonist, preferably in a γ-aminobutyric acid receptor bindingamount.

In an additional preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a phosphodiesterasetype 5 inhibitor, preferably in a phosphodiesterase type 5 inhibitingamount.

In an additional preferred embodiment of the combination aspects of thepresent invention, the second compound comprises an adenosine agonist,preferably in an adenosine agonistic amount.

In an additional preferred embodiment of the combination aspects of thepresent invention, the second compound comprises a CETP inhibitor,preferably in a CETP inhibitory amount.

A further preferred embodiment of the combination aspects of the presentinvention includes methods of treating diabetes in a mammal sufferingfrom diabetes comprising administering to said mammal an effectiveamount of a compound of formula I, a prodrug thereof or apharmaceutically acceptable salt of said compound or said prodrug and analdose reductase inhibitor, a prodrug of said aldose reductase inhibitoror a pharmaceutically acceptable salt of said aldose reductase inhibitoror said prodrug.

Another preferred embodiment of the combination aspect of the presentinvention provides methods of treating diabetes in a mammal sufferingfrom diabetes comprising administering to said mammal an effectiveamount of a compound of formula I, a prodrug thereof or apharmaceutically acceptable salt of said compound or said prodrug, and asodium hydrogen ion exchange (NHE-1) inhibitor, a prodrug of said NHE-1inhibitor or a pharmaceutically acceptable salt of said NHE-1 inhibitoror said prodrug.

Another preferred embodiment of the combination aspect of the presentinvention provides methods of treating diabetes in a mammal comprisingadministering to said mammal a compound of formula I, a prodrug thereofor a pharmaceutically acceptable salt of said compound or said prodrugand a glycogen phosphorylase inhibitor (GPI), a prodrug of said GPI or apharmaceutically acceptable salt of said GPI or said prodrug.

A further preferred embodiment of the combination aspects of the presentinvention includes methods of treating hyperglycemia in a mammalcomprising administering to said mammal a compound of formula I, aprodrug thereof or a pharmaceutically acceptable salt of said compoundor said prodrug and a glycogen phosphorylase inhibitor (GPI), a prodrugof said GPI or a pharmaceutically acceptable salt of said GPI or saidprodrug.

A further preferred embodiment of the combination aspects of the presentinvention includes methods of treating ischemia in a mammal comprisingadminstering to said mammal a compound of formula I, a prodrug thereofor a pharmaceutically acceptable salt of said compound or said prodrugand a glycogen phosphorylase inhibitor (GPI), a prodrug of said GPI or apharmaceutically acceptable salt of said GPI or said prodrug.

Another preferred embodiment of the combination aspect of the presentinvention provides methods of treating ischemia in a mammal sufferingfrom ischemia comprising administering to said mammal an effectiveamount of a compound of formula I, a prodrug thereof or apharmaceutically acceptable salt of said compound or said prodrug and asodium hydrogen ion exchange (NHE-1) inhibitor, a prodrug of said NHE-1inhibitor or a pharmaceutically acceptable salt of said NHE-1 inhibitoror said prodrug. The present invention also provides such methodswherein said ischemia is perioperative myocardial ischemia.

In addition, the present invention provides methods of reducing tissuedamage resulting from ischemia comprising administering to a mammal inneed of said treatment an effective amount of a compound of formula I, aprodrug thereof or a pharmaceutically acceptable salt of said compoundor said prodrug; wherein said ischemia is a result of an etiologyindependent of diabetic microangiopathy or diabetic macroangiopathy. Thepresent invention provides such methods wherein the tissue is heart,brain, liver, kidney, lung, gut, skeletal muscle, spleen, pancreas,retina or intestinal tissue.

In addition, the present invention provides methods of providing acardioprotective effect in a mammal which comprises administering to themammal an effective amount of a compound of formula I, a prodrug thereofor a pharmaceutically acceptable salt of said compound or said prodrug.

Also, the present invention provides processes for preparing a compoundof formula 3-8C

wherein the variables are as defined above;

which comprises the steps of:

a) reacting a compound of formula 3-5

wherein Y is a) —(C₁-C₄)alkyl, b) —C(O)—(C₁-C₄)alkyl-N—((C₁-C₄)alkyl)₂,c) —C(O)—(C₁-C₄)alkyl, d) —C(O)-phenyl or e) —CH₂-phenyl; wherein eachoccurrence of phenyl is optionally substituted with one or twosubstituents, each independently selected from —(C₁-C₄)alkyl,—O—(C₁-C₄)alkyl, fluoro or chloro; and wherein R¹ is as defined above;with an R²- and R³-substituted piperazine wherein R² and R³ are asdefined above; in a reaction inert solvent and in the presence of atertiary amine base to obtain a compound of formula 3-7

wherein the variables are as defined above;

b) reacting the compound of formula 3-7 with a compound of formula 3-8

wherein the variables are as defined above; to obtain a compound offormula 3-8A

wherein the variables are as defined above;

c) dechlorinating the compound of formula 3-8A to obtain a compound offormula 3-8B

wherein the variables are as defined above; and

d) deprotecting the compound of formula 3-8B to obtain a compound offormula 3-8C

wherein the variables are as defined above.

The present invention also provides compounds selected from the groupconsisting of:

-   2,4-dichloro-6-(1-methoxyethyl)-[1,3,5]triazine; and-   2,4-dichloro-6-(1-benzyloxyethyl)-[1,3,5]triazine.

The present invention also provides the following compounds:

compounds of formula 3-7

wherein Y is a) —(C₁-C₄)alkyl, b) —C(O)—(C₁-C₄)alkyl-N—((C₁-C₄)alkyl)₂,c) —C(O)—(C₁-C₄)alkyl, d) —C(O)-phenyl or e) —CH₂-phenyl; wherein eachoccurrence of phenyl is optionally substituted with one or twosubstituents, each independently selected from —(C₁-C₄)alkyl,—O—(C₁-C₄)alkyl, fluoro or chloro; and the other variables are asdefined above;

compounds of formula 3-8A

wherein Y is a) —(C₁-C₄)alkyl, b) —C(O)—(C₁-C₄)alkyl-N—((C₁-C₄)alkyl)₂,c) —C(O)—(C₁-C₄)alkyl, d) —C(O)-phenyl or e) —CH₂-phenyl; wherein eachoccurrence of phenyl is optionally substituted with one or twosubstituents, each independently selected from —(C₁-C₄)alkyl,—O—(C₁-C₄)alkyl, fluoro or chloro; and the other variables are asdefined above; and

compounds of formula 3-8B

wherein Y is a) —(C₁-C₄)alkyl, b) —C(O)—(C₁-C₄)alkyl-N—((C₁-C₄)alkyl)₂,c) —C(O)—(C₁-C₄)alkyl, d) —C(O)-phenyl or e) —CH₂-phenyl; wherein eachoccurrence of phenyl is optionally substituted with one or twosubstituents, each independently selected from —(C₁-C₄)alkyl,—O—(C₁-C₄)alkyl, fluoro or chloro; and the other variables are asdefined above.

The expressions “compound(s) of formula I” and “compound(s) of thepresent invention” as used herein, means a compound or compounds offormula I, isomer(s) thereof, prodrug(s) of said compound(s) orisomer(s), and pharmaceutically acceptable salt(s) of said compound(s),isomer(s) or prodrug(s). The term “compound(s),” when referring tocompounds of formula I, also includes isomer(s) of said compound(s),prodrug(s) of said compound(s) or isomer(s), and pharmaceuticallyacceptable salt(s) of said compound(s), isomer(s) or prodrug(s).

The subject invention also includes isotopically-labeled compounds,which are identical to those recited in formula I, but for the fact thatone or more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe present invention include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorous, sulfur, fluorine and chlorine, such as ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, ¹³⁵I, and ³⁶Cl, respectivelyCompounds of the present invention, which contain the aforementionedisotopes and/or other isotopes of other atoms, are within the scope ofthe present invention. Certain isotopically-labeled compounds of thepresent invention, for example, those into which radioactive isotopessuch as ³H or ¹⁴C are incorporated, are useful in drug and/or substratetissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e.,¹⁴C, isotopes are particularly preferred for their ease of preparationand detectability. Further, substitution with heavier isotopes such asdeuterium, i.e., ²H, may afford certain therapeutic advantages resultingfrom greater metabolic stability, for example increased in vivohalf-life or reduced dosage requirements and, hence, may be preferred insome circumstances. Isotopically labeled compounds of formula I of thepresent invention can generally be prepared by carrying out theprocedures disclosed in the Schemes and/or in the Examples below, bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent.

The term “reduction” is intended to include, in addition tosubstantially total prevention, partial prevention or prevention which,although greater than that which would result from taking no compound orfrom taking a placebo, is less than 100%.

The term “damage resulting from ischemia” as employed herein refers toconditions directly associated with reduced blood flow to tissue, forexample, due to a clot or obstruction of blood vessels which supplyblood to the subject tissue and which result, inter alia, in loweredoxygen transport to such tissue, impaired tissue performance, tissuedysfunction and/or necrosis. Alternatively, where blood flow or organperfusion may be quantitatively adequate, the oxygen carrying capacityof the blood or organ perfusion medium may be reduced, e.g., in anhypoxic environment, such that oxygen supply to the tissue is lowered,and impaired tissue performance, tissue dysfunction, and/or tissuenecrosis ensues.

The term “treating”, “treat” or “treatment” as used herein includespreventative (e.g., prophylactic) and palliative treatment.

The term “pharmaceutically acceptable” means the carrier, diluent,excipient, and/or salt must be compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof.

The term “effective amount” means an amount of a compound or combinationof compounds that ameliorates, attenuates or eliminates a particulardisease or condition or a symptom of a particular disease or condition,or prevents or delays the onset of a particular disease or condition ora symptom of a particular disease or condition.

The expression “prodrug” refers to a compound that is a drug precursorwhich following administration, releases the drug in vivo via somechemical or physiological process (e.g., a prodrug on being brought tothe physiological pH or through enzyme action is converted to thedesired drug form). Prodrugs of the compounds of the present inventioninclude, e.g., derivatives of the hydroxyl group in the compound offormula I wherein H is replaced by —(C₁-C₄)alkyl,—C(O)—(C₁-C₄)alkyl-N—(C₁-C₄ alkyl)₂, —C(O)—(C₁-C₄)alkyl, —C(O)-phenyl,—CH₂-phenyl in which the phenyl ring is optionally substituted with oneor two substituents selected from, e.g, —(C₁-C₄)alkyl, fluoro, chloro or—O—(C₁-C₄)alkyl.

This invention is further directed to compounds which are mutualprodrugs of aldose reductase inhibitors and sorbitol dehydrogenaseinhibitors. By mutual prodrug is meant a compound which contains twoactive components, in this case, an aldose reductase inhibitor and asorbitol dehydrogenase inhibitor, which, following administration, iscleaved, releasing each individual active component. Such mutualprodrugs of an aldose reductase inhibitor and a sorbitol dehydrogenaseinhibitor are formed under standard esterification conditions well knownto those skilled in the art. For example, mutual prodrugs of thecompounds of the present invention and an aldose reductase inhibitorwould include compounds of formula I wherein the hydrogen atom of thehydroxy group, which appears in formula I, is replaced with an acylradical of a carboxylic acid aldose reductase inhibitor. Examples ofcarboxylic acid aldose reductase inhibitors would include ponalrestat,tolrestat, zenarastat, zopolrestat and epalrestat.

By alkylene is meant an unsaturated hydrocarbon (straight chain orbranched) wherein a hydrogen atom is removed from each of the terminalcarbons. Exemplary of such groups (assuming the designated lengthencompasses the particular example) are methylene, ethylene, propylene,butylene, pentylene, hexylene and heptylene.

By halo is meant fluoro, chloro, bromo or iodo.

By alkyl is meant a saturated hydrocarbon (straight chain or branched).Exemplary of such alkyl groups (assuming the designated lengthencompasses the particular example) are methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl,neopentyl, tertiary pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl,hexyl, isohexyl, heptyl and octyl.

By cycloalkyl is meant a cyclic hydrocarbon. Examples of cycloalkylgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl. Preferred cycloalkyl groups are (C₃-C₆)cycloalkyl.

By alkoxy is meant saturated alkyl (straight chain or branched) bondedthrough an oxygen. Exemplary of such alkoxy groups (assuming thedesignated length encompasses the particular example) are methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy,pentoxy, isopentoxy, neopentoxy, tertiary pentoxy, hexoxy, isohexoxy,heptoxy and octoxy.

The term “substituted” when used to describe a phenyl or naphthyl ring,refers to replacement of a hydrogen atom of the phenyl or naphthyl ringwith another atom or group of atoms. For example, the term“mono-substituted” means that only one of the hydrogens of the phenyl ornaphthyl ring has been substituted. The term “di-substituted” means thattwo of the hydrogens of the phenyl or naphthyl ring have beensubstituted.

It is to be understood that if a carbocyclic or heterocyclic moiety maybe bonded or otherwise attached to a designated substrate throughdiffering ring atoms without denoting a specific point of attachment,then all possible points of attachment are intended, whether through acarbon atom or, for example, a trivalent nitrogen atom. For example, theterm “pyridyl” means 2-, 3- or 4-pyridyl; the term “thienyl” means 2- or3-thienyl, and so forth.

The expression “pharmaceutically acceptable salt” refers to nontoxicanionic salts containing anions such as (but not limited to) chloride,bromide, iodide, sulfate, bisulfate, phosphate, acetate, maleate,fumarate, oxalate, lactate, tartrate, citrate, gluconate,methanesulfonate and 4-toluene-sulfonate. Where more than one basicmoiety exists the expression includes multiple salts (e.g., di-salt).The expression also refers to nontoxic cationic salts such as (but notlimited to) sodium, potassium, calcium, magnesium, ammonium orprotonated benzathine (N,N′-dibenzylethylenediamine), choline,ethanolamine, diethanolamine, ethylenediamine, meglamine(N-methyl-glucamine), benethamine (N-benzylphenethylamine), piperazineor tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol).

As used herein, the expressions “reaction inert solvent” and “inertsolvent” refer to a solvent or mixture of solvents which does notinteract with starting materials, reagents, intermediates or products ina manner which adversely affects the yield of the desired product.

The chemist of ordinary skill in the art will recognize that certaincompounds of formula I of this invention will contain one or more atomswhich may be in a particular stereochemical or geometric configuration,giving rise to stereoisomers and configurational isomers. All suchisomers and mixtures thereof are included in this invention. Compoundsof formula I may be chiral. In such cases, the isomer wherein theasymmetric carbon atom attached to R¹ has the R configuration ispreferred.

Those skilled in the art will further recognize that the compounds offormula I can exist in crystalline form as hydrates wherein molecules ofwater are incorporated within the crystal structure thereof and assolvates wherein molecules of a solvent are incorporated therein. Allsuch hydrate and solvate forms are considered part of this invention.

The chemist of ordinary skill in the art will also recognize thatcertain compounds of formula I of this invention can exist in tautomericform, i.e., that an equilibrium exists between two isomers which are inrapid equilibrium with each other. A common example of tautomerism isketo-enol tautomerism, i.e.,

Examples of compounds which can exist as tautomers includehydroxypyridines, hydroxypyrmidines, hydroxyquinolines andhydroxytriazines. Other examples will be recognized by those skilled inthe art. All such tautomers and mixtures thereof are included in thisinvention.

DMF means N,N-dimethylformamide. DMSO means dimethyl sulfoxide. THFmeans tetrahydrofuran.

Whenever the structure of a cyclic radical is shown with a bond drawnfrom outside the ring to inside the ring, it will be understood by thoseof ordinary skill in the art to mean that the bond may be attached toany atom on the ring with an available site for bonding. If the cyclicradical is a bicyclic or tricyclic radical, then the bond may beattached to any atom on any of the rings with an available site forbonding. For example,

represents any or all of the following radicals:

DETAILED DESCRIPTION OF THE INVENTION

In general, the compounds of formula I of the present invention can bemade by processes analogous to those known in the chemical arts,particularly in light of the description contained herein. Certainprocesses for the preparation of the compounds of formula I of thepresent invention are provided as further features of the presentinvention and are illustrated by the following reaction schemes. Otherprocesses are described in the experimental section.

The compounds of the present invention are prepared as described in theschemes below:

Scheme 1

Compounds of the present invention of formula I, wherein the variablesare as defined in the Summary above, can be prepared from compounds offormula IA, wherein Y is, e.g., —(C₁-C₄)alkyl,—C(O)—(C₁-C₄)alkyl-N—((C₁-C₄)alkyl)₂, —C(O)—(C₁-C₄)alkyl, —C(O)-phenyl(in which the phenyl ring is optionally substituted with one or twosubstituents, each independently selected from, e.g., —(C₁-C₄)alkyl,fluoro, chloro or —O—(C₁-C₄)alkyl) or —CH₂-phenyl (in which the phenylmoiety is optionally substituted with one or two substituents, eachindependently selected from, e.g., —(C₁-C₄)alkyl, —O—(C₁-C₄)alkyl,fluoro or chloro).

When Y is —(C₁-C₄)alkyl, the compound of formula IA is reacted withboron tribromide in either chloroform or methylene chloride. Thepreferred solvent is methylene chloride. The reaction is usuallyconducted at temperatures which are between about −70° C. and about 0°C. When Y is —C(O)—(C₁-C₄)alkyl, —C(O)—(C₁-C₄)alkyl-N—((C₁-C₄)alkyl)₂ or—C(O)-phenyl in which the phenyl ring is optionally substituted asdescribed above, the compound of formula IA is reacted with eitheraqueous sodium or potassium hydroxide or concentrated HCl. The reactioncan be conducted in the presence of water-miscible organic solvents suchas alcohols, dioxane or THF. Preferred cosolvents are ethanol, methanolor THF. The temperature of the reaction ranges from room temperature toabout 80° C., and the preferred temperature is room temperature.

When Y is —CH₂-phenyl in which the phenyl moiety is optionallysubstituted as described above, the compound of formula IA ishydrogenated in the presence of platinum or palladium catalyst in analcoholic solvent or acetic acid admixed with a small amount of amineral acid, such as concentrated HCl or H₂SO₄. The preferred alcoholicsolvent is ethanol. The reaction is conducted at room temperature and atambient or pressures up to about 2.7 atm. Alternatively, when Y is—CH₂-phenyl, as described above, the compound of formula IA can bereacted with a palladium catalyst in an alcoholic solvent in thepresence of HCl gas dissolved in ether. The preferred alcoholic solventis isopropanol. The reaction is conducted at ambient pressure and thetemperature ranges from room temperature to about 100° C.

Scheme 2

In Scheme 2, compounds of formula I, wherein the variables are asdefined in the Summary above, are obtained from the compounds of formulaIA wherein Y is —(C₁-C₄)alkyl or —CH₂-phenyl in which the phenyl moietyis optionally substituted with one or two substituents, eachindependently selected from, e.g., —(C₁-C₄)alkyl, —O—(C₁-C₄)alkyl,fluoro or chloro.

Compounds of formula 2-2, wherein L¹ and L² are the same or differentand are —(C₁-C₄)alkyl, are prepared by reacting compounds of formula2-1, which are commerically available, with L¹L²NCOL³, in which L³ ischloro, in ether or halocarbon solvents such as ether, THF, chloroformor methylene chloride. The preferred solvents are THF and methylenechloride. The reaction is conducted in the presence of a tertiarynitrogen base such as triethylamine, dimethylisopropylamine or pyridine.The reaction temperature is between room temperature and about 100° C.The preferred temperature is room temperature. Compounds of formula 2-3are prepared by reacting compounds of formula 2-2 with phosphorustrichloride, phosphorus pentachloride or phosphorus oxychloride at atemperature which is between about 100° C. and about 150° C.

Compounds of formula 2-5 are obtained by reacting compounds of formula2-4, which are commerically available or prepared as described inInternational Publication WO 00/59510, published 12 Oct. 2000, withcyanogen bromide in acetonitrile admixed with an alcoholic solvent suchas ethanol or isopropanol. The preferred alcoholic solvent is ethanol.The reaction is conducted in the temperature range of about 0° C. toabout 40° C. In Step 4 of Scheme 2, compounds of formula 2-3 are reactedwith compounds of formula 2-5 in acetonitrile, according to theprocedures set forth in Synthesis, 1980, 841-842, to give compounds offormula 2-6.

In Step 5 of Scheme 2, compounds of formula 2-6 are hydrogenated toremove the chlorine atom and to obtain compounds of formula IA. Thisreaction is conducted in the presence of hydrogen, either palladium orplatinum catalyst, and an alcoholic solvent such as ethanol, containingsodium or potassium hydroxide. The reaction is conducted at highpressure in the range of about 2.7 to about 3.4 atm. In Step 6 of Scheme2, the compounds of formula I are obtained from compounds of formula IA,according to procedures described in Scheme 1 above.

Scheme 3

Scheme 3 discloses alternative procedures for the preparation ofcompounds of formula I, wherein the variables are as defined in theSummary above, from the compounds of formula IA wherein Y is—(C₁-C₄)alkyl or —CH₂-phenyl in which the phenyl moiety is optionallysubstituted with one or two substituents, each independently selectedfrom, e.g., —(C₁-C₄)alkyl, —O—(C₁-C₄)alkyl, fluoro or chloro.

General procedures for the preparation of compounds of formula 3-1,wherein Y is defined above and R¹ is defined in the Summary above, aredescribed in the literature, for example, Helv. Chim. Acta, 1971,845-851. Compounds of formula 3-1 are reacted in Step 1 withchlorosulfonyl isocyanate to obtain compounds of formula 3-2. Thereaction is conducted in a reaction-inert solvent such as ether, THF,diglyme or acetonitrile. The preferred solvent is acetonitrile. Therange of the reaction temperature is between room temperature and about60° C. The reaction is conducted at ambient pressure. Compounds offormula 3-2 are reacted in Step 2 with chlorosulfonyl isocyanate toobtain compounds of formula 3-3. The reaction is conducted in areaction-inert solvent such as ether, THF, diglyme or acetonitrile. Thepreferred solvent is acetonitrile. The reaction temperature is betweenroom temperature and about 60° C. The reaction is conducted at ambientpressure.

Compounds of formula 3-3 are cyclized in Step 3 to give compounds offormula 3-4, using either aqueous NaOH or KOH. The reaction is conductedat temperatures which are between about 0° C. and room temperature andat ambient pressure. Compounds of formula 3-5 are prepared in Step 4 byreacting compounds of formula 3-4 with PCl₃, PCl₅ or POCl₃ in thepresence of a tertiary nitrogen base such as a trialkyl amine or adialkyl aniline. The preferred conditions are POCl₃ in the presence ofdiethyl aniline. The reaction is usually conducted at temperatures whichare between about 50° and about 100° C. and at ambient pressure. Step 5involves the displacement of one of the chlorine atoms in the compoundsof formula 3-5 by substituted piperazines having substituents R², R³ andX as described in the Summary above, in the presence of either aninorganic base, such as sodium or potassium bicarbonate or carbonate, ora tertiary nitrogen base, such as triethylamine, isopropylethylamine ordiazabicyclononane, to obtain compounds of formula 3-6. The preferredbases are sodium or potassium bicarbonate. The reaction is conducted atambient pressure, at temperatures which are between room temperature andabout 60° C., and in a polar non-aqueous solvent, such as acetonitrileor DMF. The preferred temperature is room temperature and the preferredsolvent is DMF.

In Step 6, compounds of formula 3-6 are hydrogenated to remove thechlorine atom to give the compounds of formula IA. This reaction isconducted in the presence of hydrogen, either palladium or platinumcatalyst, and an alcoholic solvent, such as ethanol, containing sodiumor potassium hydroxide. The reaction is conducted at a pressure in therange of about 2.7 to about 3.4 atm. In Step 7 of Scheme 3, thecompounds of formula I are obtained from compounds of formula IA,according to procedures described in Scheme 1 above.

Scheme 3A

Scheme 3A describes the preparation of compounds of formula I wherein Xis 1,3,5-triazine (also known as s-triazine) optionally substituted withR^(z1) and R^(z2), and the other variables are as defined in the Summaryabove.

Compounds of formula 3-5, prepared as described in Scheme 3, are reactedwith R²- and R³-substituted piperazines to obtain compounds of formula3-7. The reaction is conducted in a reaction-inert solvent such asmethylene chloride, ether, or THF and in the presence of a tertiaryamine base such as triethylamine, pyridine or an excess quantity of R²-and R³-substituted piperazine. The reaction temperature is between about0° C. and about 100° C.

Compounds of formula 3-7 are reacted with chloro-substituted-s-triazinesof formula 3-8 wherein R^(z1) and R^(z2) are as defined above, which arecommerically available or which can be made using known procedures, toobtain compounds of formula 3-8A. The compounds of formula 3-8B and 3-8Cwherein the variables are as defined in the Summary above are obtainedfrom the compounds of formula 3-8A, according to procedures described inScheme 1 above.

Scheme 3B

Compounds of formula I wherein X is s-triazine with one substituentR^(z2), and the other variables are as defined in the Summary above, areprepared as described in Scheme 3B.

Compounds of formula 3-7, prepared as described in Scheme 3A, arereacted with boron tribromide in either chloroform or methylene chlorideto give compounds of formula 3-9. The preferred solvent is methylenechloride. The reaction is usually conducted at temperatures which arebetween about −70° C. and about 0° C. Compounds of formula 3-9 arereacted with dichloro-substituted-s-triazine of formula 3-10, which arecommercially available, to obtain compounds of formula 3-11. Thereaction is conducted in the presence of a tertiary amine base, such astriethylamine, pyridine or excess compound of formula 3-7 in areaction-inert solvent, such as methylene chloride, chloroform or THF.

Reductive removal of the chlorine atoms in the compounds of formula 3-11completes the sequence of steps to obtain the compounds of formula 3-11A wherein the variables are as defined in the Summary above. Thereaction is catalyzed by palladium or platinum catalysts, in a Parrhydrogenator at pressures in the range of about 3.1 to about 3.7 atm andat room temperature. Solvents for the reaction are alcoholic solventsand the preferred alcohol is isopropanol.

Scheme 3C

Alternatively, compounds of formula I wherein X is s-triazine with onesubstituent, hydroxymethyl-, and the other variables are as defined inthe Summary above, can be prepared from the compounds of formula 3-9, asdescribed in Scheme 3C.

Compounds of formula 3-9, prepared as described in Scheme 3B, arereacted with compounds of formula 3-12, prepared as described inJ.Amer.Chem.Soc., 79 (1957) 944-948, and J.Chem.Soc., (1958) 1134-1139,in the presence of a tertiary amine base, such as triethylamine,pyridine or an excess quantity of the compound of formula 3-9, in areaction-inert solvent, such as methylene chloride, chloroform or THF,to obtain compounds of formula 3-13. The reaction temperature is betweenabout 0° C. and the refluxing temperature of the solvent used.

Compounds of formula 3-13 are reacted with sulfuric acid in areaction-inert solvent, such as EtOAc, dioxane, ether or THF, to obtaincompounds of formula 3-14. Reductive removal of the chlorine atoms incompounds of formula 3-14 completes the sequence of steps in thepreparation of compounds of formula 3-14A, wherein the variables are asdefined in the Summary above. The reaction is catalyzed by palladium orplatinum catalysts, in a Parr hydrogenator at pressures in the range ofabout 3.1 to about 3.7 atm and at room temperature. Solvents for thereaction are alcoholic solvents and the preferred alcohol isisopropanol.

Scheme 3D

Alternatively, compounds of formula I, wherein R¹ is hydrogen, R² and R³are as described in the Summary above, and X is s-triazine havingsubstituents R^(z1) and R^(z2), can be prepared according to theprocedures set forth in Scheme 3D. For example, compounds of formula I,wherein X is s-triazine, R^(z1) is methyl and R^(z2) is phenyloptionally substituted as described in the Summary above, can also beprepared by procedures set forth in Scheme 3D.

Compounds of formula 3-15 are prepared by reacting R^(z2)—CN withtrichloroacetonitrile and aluminum bromide in the presence of HCl gas.The reaction is conducted at temperatures which are between about 0° C.and about 20° C. and at ambient pressure. Compounds of formula 3-16 areprepared by reacting compounds of formula 3-15 with piperazines havingR² and R³ substituents as defined in the Summary above. The reaction isconducted in a reaction-inert solvent, such as methylene chloride, etheror THF, and in the presence of a tertiary amine base, such astriethylamine, pyridine or an excess quantity of R² and R³-substitutedpiperazine. The reaction temperature is between about 0° C. and about100° C.

Compounds of formula 3-17 are prepared by hydrogenating compounds offormula 3-16 to remove the chlorine atoms. This reaction is conducted inthe presence of hydrogen, either palladium or platinum catalysts, and analcoholic solvent, such as ethanol, containing sodium or potassiumhydroxide. The reaction is conducted at a pressure in the range of about2.7 to about 3.4 atm. Compounds of formula 3-18 are prepared by reactingcompounds of formula 3-17 with 2,4-dichloro-6-diazomethyl-triazine, thecompound of formula 3-12, which is also used in Scheme 3C above. Thisreaction is conducted in a reaction-inert solvent, such as methylenechloride, ether or THF, and in the presence of a tertiary amine base,such as triethylamine or pyridine. The reaction temperature is betweenabout 0° C. and about 100° C.

Compounds of formula 3-19 are prepared by reacting compounds of formula3-18 with sulfuric acid in the presence of a reaction-inert solvent,such as ethyl acetate, ether or THF. The reaction temperature is betweenabout 0° C. and about 100° C. Compounds of formula 3-19 are transformedto compounds of formula 3-20 by removing the chlorine atoms throughhydrogenation mediated by catalysts, such as palladium or platinum, inthe presence of sodium or potassium hydroxide. The reaction is conductedat a pressure in the range of about 2.7 to about 3.4 atm and understandard reaction conditions.

Scheme 3E

Compounds of formula I wherein R¹, R² and R³ are as described in theSummary above and X is —C(O)—R⁴-Z, —SO₂—R⁴-Z, —C(O)—NR⁵R⁶ or —SO₂—NR⁵R⁶,are prepared as described in Scheme 3E.

Compounds of formula 3-7, prepared as described in Scheme 3A, arereacted with L⁴-C(O)—R⁴-Z, L⁴-SO₂—R⁴-Z, L⁴-CO—NR⁵R⁶ or L⁴-SO₂—NR⁵R⁶,wherein L⁴ is, e.g., chloro and the variables are as defined in theSummary above, in the presence of a tertiary amine base, such astriethylamine, pyridine or an excess quantity of the compound of formula3-7, in a reaction-inert solvent, such as methylene chloride, chloroformor THF, to give the compound of formula IA. The reaction temperature isbetween about 0° C. and the refluxing temperature of the solvent used.The compounds of formula I are obtained from compounds of formula IA,according to procedures described in Scheme 1 above.

The starting materials and reagents for the above described compoundsare also readily available or can be easily synthesized by those skilledin the art using conventional methods of organic synthesis. For example,many of the compounds used herein are related to, or are derived from,compounds found in nature, in which there is great scientific interestand commercial need, and accordingly many such compounds arecommercially available or are reported in the literature or are easilyprepared from other commonly available substances by methods which arereported in the literature.

The compounds of the present invention inhibit the enzyme activity ofsorbitol dehydrogenase and as such have utility in the treatment ofdiabetic complications including, but not limited to, complications suchas diabetic nephropathy, diabetic neuropathy, diabetic retinopathy,diabetic microangiopathy, diabetic macroangiopathy, diabeticcardiomyopathy and foot ulcers, in mammals. The compounds of the presentinvention also have utility in providing a cardioprotective effect inmammals.

The utility of the compounds of the present invention as medical agentsin the treatment of diseases, such as are detailed herein, in mammals(e.g., humans) is demonstrated by the activity of the compounds offormula I of this invention in conventional assays. Such assays alsoprovide a means whereby the activities of the compounds of formula I ofthe present invention can be compared with the activities of other knowncompounds. The results of these comparisons are useful for determiningdosage levels in mammals, including humans, for the treatment of suchdiseases.

Measurement of SDH Activity

Male Sprague-Dawley rats (200-250 g) are used for these experiments.Diabetes is induced in some of the rats by a tail vein injection ofstreptozocin, 85 mg/kg. Twenty-four hours later, 4 groups of diabeticrats are given a single dose of the test compound (0.001 to 100 mg/kg)by oral gavage. Animals are sacrificed 4-6 hours after dosing and bloodand sciatic nerves are harvested. Tissues and cells are extracted with6% perchloric acid.

Sorbitol in erythrocytes and nerves is measured by a modification of themethod of R. S. Clements et al. (Science, 166: 1007-8, 1969). Aliquotsof tissue extracts are added to an assay system which has finalconcentrations of reagents of 0.033 M glycine, pH 9.4, 800 mM B-nicotineadenine dinucleotide, and 4 units/ml of sorbitol dehydrogenase. Afterincubation for 30 minutes at room temperature, sample fluorescence isdetermined on a fluorescence spectrophotometer with excitation at 366 nmand emission at 452 nm. After subtracting appropriate blanks, the amountof sorbitol in each sample is determined from a linear regression ofsorbitol standards processed in the same manner as the tissue extracts.

Fructose is determined by a modification of the method described by M.Ameyama, Methods in Enzymology, 89: 20-25 (1982). Resazurin issubstituted for ferricyamide. Aliquots of tissue extracts are added tothe assay system, which has final concentrations of reagents of 1.2 Mcitric acid, pH 4.5, 13 mM resazurin, 3.3 units/ml of fructosedehydrogenase and 0.068% Triton X-100. After incubation for 60 minutesat room temperature, sample fluorescence is determined on a fluorescencespectrophotometer with excitation at 560 nm and emission at 580 nm.After subtracting appropriate blanks, the amount of fructose in eachsample is determined from a linear regression of fructose standardsprocessed in the same manner as the tissue extracts.

SDH activity is measured by a modification of the method described by U.Gerlach, Methodology of Enzymatic Analyses, edited by H. U. Bergmeyer,3, 112-117 (1983). Aliquots of sera or urine are added to the assaysystem, which has final concentrations of reagents of 0.1 M potassiumphosphate buffer, pH 7.4, 5 mM NAD, 20 mM sorbitol, and 0.7 units/ml ofsorbitol dehydrogenase. After incubation for 10 minutes at roomtemperature, the average change in sample absorbance is determined at340 nm. SDH activity is presented as milliOD₃₄₀ units/minute(OD₃₄₀=optical density at 340 nm).

The activity and thus utility of the compounds of the present inventionas medical agents in providing protection from ischemic damage to tissuein a mammal can be demonstrated by the activity of the compounds in thein vitro assay described below and in U.S. Pat. No. 5,932,581, which ishereby incorporated by reference herein. This assay is more particularlydirected to providing protection from ischemic damage to myocardialtissue (e.g., for inducing cardioprotection). The assay also provides ameans whereby the activities of the compounds of the present inventioncan be compared with the activities of other known compounds. Theresults of these comparisons are useful for determining dosage levels inmammals, including humans, for inducing protection from ischemia,particularly in the myocardium.

Cardioprotection, as indicated by a reduction in infarcted myocardium,can be induced pharmacologically using adenosine receptor agonists inisolated, retrogradely perfused rabbit hearts as an in vitro model ofmyocardial ischemic preconditioning (Liu et al., Cardiovasc. Res.,28:1057-1061, 1994; and Tracey et al., Cardiovasc. Res., 28:410-415,1997). The in vitro test described below demonstrates that a testcompound (i.e., a compound of formula I of the present invention) canalso pharmacologically induce cardioprotection, i.e., reduced myocardialinfarct size, when administered to a rabbit isolated heart. The effectsof the test compound are compared to ischemic preconditioning and theA1/A3 adenosine agonist, APNEA (N⁶-[2-(4-aminophenyl)ethyl]adenosine),that has been shown to pharmacologically induce cardioprotection in therabbit isolated heart (Liu et al., Cardiovasc. Res., 28:1057-1061,1994). The exact methodology is described below:

The protocol used for these experiments closely follows that describedby Liu et al., Cardiovasc. Res., 28:1057-1061, 1994. Male New ZealandWhite rabbits (3-4 kg) are anesthetized with sodium pentobarbital (30mg/kg, i.v.). After deep anesthesia is achieved (determined by theabsence of an ocular blink reflex) the animal is intubated andventilated with 100% O₂ using a positive pressure ventilator. A leftthoracotomy is performed, the heart exposed, and a snare (2-0 silk) isplaced loosely around a branch of the left anterior descending coronaryartery, approximately ⅔ of the distance towards the apex of the heart.The heart is removed from the chest and rapidly (<30 cc) mounted on aLangendorff apparatus. The heart is retrogradely used via the aorta in anon-recirculating manner with a modified Krebs solution (NaCl 118.5 mM,KCl 4.7 mM, Mg SO₄ 1.2 mM, KH₂PO₄ 1.2 mM, NaHCO₃ 24.8 mM, CaCl₂ 2.5 mM,and glucose 10 mM), at a constant pressure of 80 mmHg and a temperatureof 37° C. Perfusate pH is maintained at 7.4-7.5 by bubbling with 95%O₂/5% CO₂. Heart temperature is tightly controlled by using heatedreservoirs for the physiological solution and water jacketing aroundboth the perfusion tubing and the isolated heart. Heart rate and leftventricular pressures are determined via a latex balloon which isinserted in the left ventricle and connected by stainless steel tubingto a pressure transducer. The intraventricular balloon is inflated toprovide a systolic pressure of 80-100 mmHg, and a diastolic pressureless than or equal to 10 mmHg. Perfusate flow rates are routinelydetermined throughout the experimental period.

The heart is allowed to equilibrate for 30 min, over which time theheart must show stable left ventricular pressures within the parametersoutlined above. If the heart rate falls below 180 bpm at any time priorto the 30 min period of regional ischemia, the heart is paced at ≈200bpm for the remainder of the experiment. ischemic preconditioning isinduced by total cessation of cardiac perfusion (global ischemia) for 5min, followed by reperfusion for 10 min. The global ischemia/reperfusionis repeated one additional time, followed by a 30 min regional ischemia.The regional ischemia is provided by tightening the snare around thecoronary artery branch. Following the 30 min regional ischemia, thesnare is released and the heart reperfused for an additional 120 min.

Pharmacological cardioprotection is induced by infusing the testcompound at predetermined concentrations, starting 30 min prior to the30 min regional ischemia, and continuing until the end of the 120 minreperfusion period. Hearts which receive test compounds do not undergothe two periods of ischemic preconditioning. The reference compound,APNEA (500 nM) is perfused through hearts (which do not receive the testcompound) for a 5 min period which ends 10 min before the 30 minregional ischemia.

At the end of the 120 min reperfusion period, the coronary artery snareis tightened, and a 0.5% suspension of fluorescent zinc cadmium sulfateparticles (1-10 μM) is perfused through the heart; this stains all ofthe myocardium, except that area at risk for infarct development(area-at-risk). The heart is removed from the Langendorff apparatus,blotted dry, weighed, wrapped in aluminum foil and stored overnight at−20° C. The next day, the heart is sliced into 2 mm transverse sectionsfrom the apex to just above the coronary artery snare. The slices arestained with 1% triphenyl tetrazolium chloride (TTC) inphosphate-buffered saline for 20 min at 37° C. Since TTC reacts withliving tissue (containing NAD-dependent dehydrogenases), this staindifferentiates between living (red stained) tissue, and dead tissue(unstained infarcted tissue). The infarcted area (no stain) and thearea-at-risk (no fluorescent particles) are calculated for each slice ofleft ventricle using a precalibrated image analyzer. To normalize theischemic injury for difference in the area-at-risk between hearts, thedata is expressed as the ratio of infarct area vs. area-at-risk(%IA/AR).

The activity and thus utility of the compounds of the present inventionas medical agents in providing protection from ischemic damage to tissuein a mammal can be further demonstrated by the activity of the compoundsin the in vitro assay described below. The assay also provides a meanswhereby the activities of the compounds of the present invention can becompared with the activities of other known compounds. The results ofthese comparisons are useful for determining dosage levels in mammals,including humans, for inducing protection from ischemia.

The activity of a sorbitol dehydrogenase inhibitor in a tissue can bedetermined by testing the amount of sorbitol dehydrogenase inhibitorthat is required to raise tissue sorbitol (i.e., by inhibiting thefurther metabolism of sorbitol consequent to blocking sorbitoldehydrogenase) or lower tissue fructose (by inhibiting its productionfrom sorbitol consequent to blocking sorbitol dehydrogenase). While notwishing to be bound by any particular theory or mechanism, it isbelieved that a sorbitol dehydrogenase inhibitor, by inhibiting sorbitoldehydrogenase, prevents or reduces ischemic damage as describedhereinafter in the following paragraph and scheme, appearing at column 9of U.S. Pat. No. 5,932,581, which is hereby incorporated by referenceherein.

When the supply of oxygenated blood to a tissue is interrupted or sloweddown (ischemia) the cells in the oxygen-deficient tissue derive theirenergy (ATP) from glucose via glycolysis (which does not require thepresence of oxygen). Glycolysis also requires a supply of NAD⁺ and in anischemic tissue the length of time glycolysis can be maintained becomessensitive to the supply of NAD⁺. However, sorbitol dehydrogenase (SDH)also utilizes NAD⁺ but does not produce an increase in ATP. Thus, itfollows that preventing or retarding NAD⁺ use by SDH with sorbitoldehydrogenase inhibitors (SDIs) will enhance or prolong the ability ofischemic tissue to carry out glycolysis, i.e., to produce energy in theabsence of oxygen and in turn enhance and prolong the survival of thecells in the tissue. Since inhibition of SDH will retard depletion ofthe tissue's NAD⁺, a sorbitol dehydrogenase inhibitor is an effectiveanti-ischemic agent.

Again, the activity of a sorbitol dehydrogenase inhibitor can bedetermined by the amount of sorbitol dehydrogenase inhibitor that isrequired to raise tissue sorbitol or lower tissue fructose. MaleSprague-Dawley rats are rendered diabetic by injection of streptozocinat 55 mg/kg, i.v., in pH 4.5 citrate buffer. They are fed ad libitum incontrolled conditions of housing, temperature and lighting. After fiveweeks of diabetes, the rats are anesthetized with an overdose ofpentobarbital, and tissues are rapidly removed and analyzed for sorbitoland fructose. Sorbitol levels are analyzed according to the method ofDonald M. Eades et al., “Rapid Analysis of Sorbitol, Galactitol,Mannitol and Myoinositol Mixtures From Biological Sources,” Journal ofChromatography, 490, 1-8, (1989).

Fructose in rat tissues is enzymatically measured using a modificationof the method of Ameyama (Methods in Enzymology, 89:20-29 1982), inwhich ferricyamide was replaced by resazurin, a dye that is reduced tothe highly fluorescent resorufin. The amount of resorufin fluorescenceis stoichiometric with the amount of fructose oxidized by fructosedehydrogenase. The assay contains 0.1 ml neutralized 6% perchloric acidnerve extract in a final volume of 1.5 ml. Following incubation for 60minutes at room temperature in a closed drawer, sample fluorescence isdetermined at excitation=560 nm, emission=580 nm with slits of 5 mm eachon a Perkin-Elmer model 650-40 fluorescence spectrophotometer. Fructoseconcentrations are calculated by comparison with a series of knownfructose standards.

The sorbitol dehydrogenase inhibitor compounds of the present inventionare thus useful in reducing or minimizing damage effected directly toany tissue that may be susceptible to ischemia/reperfusion injury (e.g.,heart, brain, lung, kidney, liver, gut, skeletal muscle, retina) as theresult of an ischemic event (e.g., myocardial infarction). A compound ofthe present invention is therefore usefully employed prophylactically toprevent, i.e. (prospectively or prophylactically) to blunt or stem,tissue damage (e.g., myocardial tissue) in patients who are at risk forischemia (e.g., myocardial ischemia).

The sorbitol dehydrogenase inhibitor compounds of the present inventionare particularly well suited to the treatment of diabetic patientsbecause of increased metabolism through sorbitol dehydrogenase in thediabetic state. The compounds of the present invention are also wellsuited for prophylactic use with non-diabetic patients who have actuallysuffered or who are considered at risk of suffering from ischemic events(e.g., myocardial ischemia).

The compounds of formula I of the present invention may be administeredto a subject in need of treatment by a variety of conventional routes ofadministration, including orally, parenterally, topically and rectally,as described further below. In general, compounds of formula I and theirpharmaceutically acceptable salts will be administered orally orparenterally at dosages between about 0.001 and about 100 mg/kg bodyweight of the subject to be treated per day, preferably from about 0.01to 10 mg/kg, in single or divided doses. However, some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The individual responsible for dosing will, in any event,determine the appropriate dose for the individual subject.

Mutual prodrugs of compounds of formula I and aldose reductaseinhibitors, as described below, will generally be administered orally orparenterally at dosages between about 0.001 and about 100 mg/kg bodyweight of the subject to be treated per day, preferably from about 0.01to about 10 mg/kg, in single or divided doses. Compositions containingboth a compound of the formula I and an aldose reductase inhibitor willgenerally be administered orally or parenterally at dosages betweenabout 0.001 and about 100 mg of each active component (i.e., thecompound of formula I and the aldose reductase inhibitor) per kg bodyweight of the subject to be treated per day, preferably from about 0.01to about 10 mg/kg.

The following compounds of the present invention are preferred:

4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-piperazine-1-sulfonic aciddimethylamide;

1-{4-[3R,5S-dimethyl-4-(4-methyl-[1,3,5]triazin-2-yl)-piperazin-1-yl]-[1,3,5]triazin-2-yl}-R-ethanol;

1-{4-[4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;

2-{4-[4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-6-methyl-[1,3,5]triazin-2-yl}-phenol;

1-{4-[4-(4-cyclopropyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;

dimethylamino-acetic acid1-{4-[3R,5S-dimethyl-4-(4-methyl-[1,3,5]triazin-2-yl)-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethylester;

4-[4-(1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-piperazine-1-sulfonic aciddimethylamide;

1-(4-{4-[4-(1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-[1,3,5]triazin-2-yl)-ethanol;

1-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-2R,6S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;

1-{4-[4-(4-hydroxy-3-methyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;

1-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;

1-{4-[4-(4-methoxymethy-[1,3,5]triazin-2-yl)-2R,6S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;

1-{4-[4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;

1-{4-[4-(4-methoxy-6-methyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;

1-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;

1-{4-[4-(4-hydroxy-6-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;

benzofuran-2-yl-{4-[4-1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-methanone;and

furo[2,3-c]pyridin-2-yl-{4-[4-1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-methanone.

The term “Second Agents” hereinafter refers collectively topharmaceutical compounds or agents that are aldose reductase inhibitors,sodium hydrogen ion exchange (NHE-1) inhibitors, glycogen phosphorylaseinhibitors, selective serotonin reuptake inhibitors,3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, angiotensinconverting enzyme inhibitors, thiazolidinedione antidiabetic agents,angiotensin II receptor antagonists, γ-aminobutyric acid (GABA) agonist,phosphodiesterase type 5 inhibitors or CETP inhibitors, a prodrug ofsaid compounds or agents, or a pharmaceutically acceptable salt of suchcompounds, agents or prodrugs. Use of the term in singular form, as in“a Second Agent” hereinafter refers to a pharmaceutical agent selectedfrom said Second Agents. A Second Agent may be a pharmaceutical agentthat shares more than one of the foregoing characteristics.

An additional aspect of this invention relates to pharmaceuticalcompositions comprising a compound of formula I of the presentinvention, and a Second Agent. Such compositions are hereinafterreferred to collectively as the “combination compositions”.

This invention also relates to therapeutic methods for treating orpreventing diabetic complications in a mammal wherein a compound offormula I of the present invention and a Second Agent are administeredtogether as part of the same pharmaceutical composition or separately.Such methods are hereinafter referred to collectively as the“combination therapies” of the present invention. Combination therapiesinclude therapeutic methods wherein a compound of formula I of thepresent invention and a Second Agent are administered together as partof the same pharmaceutical composition and to methods wherein these twoagents are administered separately, either simultaneously orsequentially in any order.

This invention further provides pharmaceutical kits comprising acompound of formula I of the present invention and a Second Agent. Suchkits may hereinafter be referred to as the “kits” of the presentinvention.

Any aldose reductase inhibitor may be used as the Second Agent in thecombination compositions, combination therapies and kits of the presentinvention. The term aldose reductase inhibitor refers to compounds whichinhibit the bioconversion of glucose to sorbitol catalyzed by the enzymealdose reductase. Such inhibition is readily determined by those skilledin the art according to standard assays (J. Malone, Diabetes,29:861-864, 1980. “Red Cell Sorbitol, an Indicator of DiabeticControl”). A variety of aldose reductase inhibitors are described andreferenced below; however, other aldose reductase inhibitors will beknown to those skilled in the art. The disclosures of U.S. patentslisted below are hereby incorporated by reference. Also, common chemicalUSAN names or other designation are in parentheses where applicable,together with reference to appropriate patent literature disclosing thecompound.

The activity of an aldose reductase inhibitor in a tissue can bedetermined by testing the amount of aldose reductase inhibitor that isrequired to lower tissue sorbitol (i.e., by inhibiting the furtherproduction of sorbitol consequent to blocking aldose reductase) or lowertissue fructose (by inhibiting the production of sorbitol consequent toblocking aldose reductase and consequently the production of fructose).

Accordingly, examples of aldose reductase inhibitors useful in thecompositions and methods of this invention include:

1. 3-(4-bromo-2-fluorobenzyl)-3,4-dihydro-4-oxo-1-phthalazineacetic acid(ponalrestat, U.S. Pat. No. 4,251,528);

2.N[[(5-trifluoromethyl)-6-methoxy-1-naphthalenyl]thioxomethyl]-N-methylglycine(tolrestat, U.S. Pat. No. 4,600,724);

3. 5-[(Z,E)-β-methylcinnamylidene]-4-oxo-2-thioxo-3-thiazolideneaceticacid (epalrestat, U.S. Pat. Nos. 4,464,382, 4,791,126, 4,831,045);

4. 3-(4-bromo-2-fluorobenzyl)-7-chloro-3,4-dihydro-2,4-dioxo-1(2H)-quinazolineacetic acid (zenarestat, U.S. Pat. Nos. 4,734,419, and4,883,800);

5. 2R,4R-6,7-dichloro-4-hydroxy-2-methylchroman-4-acetic acid (U.S. Pat.No. 4,883,410);

6. 2R,4R-6,7-dichloro-6-fluoro-4-hydroxy-2-methylchroman-4-acetic acid(U.S. Pat. No. 4,883,410);

7. 3,4-dihydro-2,8-diisopropyl-3-oxo-2H-1,4-benzoxazine-4-acetic acid(U.S. Pat. No. 4,771,050);

8.3,4-dihydro-3-oxo-4-[(4,5,7-trifluoro-2-benzothiazolyl)methyl]-2H-1,4-benzothiazine-2-aceticacid (SPR-210, U.S. Pat. No. 5,252,572);

9.N-[3,5-dimethyl-4-[(nitromethyl)sulfonyl]phenyl]-2-methyl-benzeneacetamide(ZD5522, U.S. Pat. Nos. 5,270,342 and 5,430,060);

10. (S)-6-fluorospiro[chroman-4,4′-imidazolidine]-2,5′-dione (sorbinil,U.S. Pat. No. 4,130,714);

11. d-2-methyl-6-fluoro-spiro(chroman-4′,4′-imidazolidine)-2′,5′-dione(U.S. Pat. No. 4,540,704);

12. 2-fluoro-spiro(9H-fluorene-9,4′-imidazolidine)-2′,5′-dione (U.S.Pat. No. 4,438,272);

13. 2,7-di-fluoro-spiro(9H-fluorene-9,4′-imidazolidine)-2′,5′-dione(U.S. Pat. Nos. 4,436,745, 4,438,272);

14.2,7-di-fluoro-5-methoxy-spiro(9H-fluorene-9,4′-imidazolidine)-2′,5′-dione(U.S. Pat. Nos. 4,436,745, 4,438,272);

15.7-fluoro-spiro(5H-indenol[1,2-b]pyridine-5,3′-pyrrolidine)-2,5′-dione(U.S. Pat. Nos. 4,436,745, 4,438,272);

16.d-cis-6′-chloro-2′,3′-dihydro-2′-methyl-spiro-(imidazolidine-4,4′-4′H-pyrano(2,3-b)pyridine)-2,5-dione (U.S. Pat. No. 4,980,357);

17.spiro[imidazolidine-4,5′(6H)-quinoline]-2,5-dione-3′-chloro-7′,8′-dihydro-7′-methyl-(5′-cis)(U.S. Pat. No. 5,066,659);

18.(2S,4S)-6-fluoro-2′,5′-dioxospiro(chroman-4,4′-imidazolidine)-2-carboxamide(U.S. Pat. No. 5,447,946); and

19. 2-[(4-bromo-2-fluorophenyl)methyl]-6-fluorospiro[isoquinoline-4(1H),3′-pyrrolidine]-1,2′,3,5′(2H)-tetrone (ARI-509, U.S. Pat. No.5,037,831).

Other aldose reductase inhibitors include compounds such as thosedisclosed in U.S. Pat. No. 4,939,140, and having the formula ARI,

or a pharmaceutically acceptable salt thereof, wherein

Z in the compounds of formula ARI is O or S;

R¹ in the compounds of formula ARI is hydroxy or a group capable ofbeing removed in vivo to produce a compound of formula ARI wherein R¹ isOH; and

X and Y in the compounds of formula ARI are the same or different andare selected from hydrogen, trifluoromethyl, fluoro, and chloro.

A preferred subgroup within the above group of aldose reductaseinhibitors includes numbered compounds 1, 2, 3, 4, 5, 6, 9, 10, and 17,and the following compounds of Formula ARI:

20.3,4-dihydro-3-(5-fluorobenzothiazol-2-ylmethyl)-4-oxophthalazin-1-yl-aceticacid [R¹=hydroxy; X=F; Y=H];

21.3-(5,7-difluorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X=Y=F];

22.3-(5-chlorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X=Cl; Y=H];

23.3-(5,7-dichlorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X=Y=Cl];

24.3,4-dihydro-4-oxo-3-(5-trifluoromethylbenzoxazol-2-ylmethyl)phthalazin-1-ylaceticacid [R¹=hydroxy; X=CF₃; Y=H];

25.3,4-dihydro-3-(5-fluorobenzoxazol-2-ylmethyl)-4-oxophthalazin-1-yl-aceticacid [R¹=hydroxy; X=F; Y=H];

26.3-(5,7-difluorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X=Y=F];

27.3-(5-chlorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X=Cl; Y=H];

28.3-(5,7-dichlorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylaceticacid [R¹=hydroxy; X=Y=Cl]; and

29. zopolrestat; 1-phthalazineacetic acid,3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl]methyl]-[R¹=hydroxy;X=trifluoromethyl; Y=H].

In compounds 20-23, and 29, Z is S. In compounds 24-28, Z is O.

Of the above subgroup, compounds 20-29 are more preferred with 29especially preferred.

An especially preferred aldose reductase inhibitor is1-phthalazineacetic acid,3,4-dihydro-4-oxo-3-[[5-trifluoromethyl)-2-benzothiazolyl]methyl]-.

The term “acyl radical of a carboxylic acid aldose reductase inhibitor”refers to any of the above-mentioned aldose reductase inhibitors whichcontains a carboxylic acid group in which the carboxylic acid group isreplaced with a carbonyl radical.

The aldose reductase inhibitor compounds of this invention are readilyavailable or can be easily synthesized by those skilled in the art usingconventional methods of organic synthesis, particularly in view of thepertinent patent specification descriptions.

An amount of the aldose reductase inhibitor of this invention that iseffective for the uses of the present invention may be used. Typically,an effective dosage for the aldose reductase inhibitors of thisinvention is in the range of about 0.1 mg/kg/day to 100 mg/kg/day insingle or divided doses, preferably 0.1 mg/kg/day to 20 mg/kg/day insingle or divided doses.

Any sodium hydrogen ion exchange (NHE-1) inhibitor may be used as theSecond Agent in the combination compositions, combination therapies andkits of the present invention. The term NHE-1 inhibitor refers tocompounds which inhibit the sodium/proton (Na⁺/H⁺) exchange transportsystem and hence are useful as a therapeutic or prophylactic agent fordiseases caused or aggravated by the acceleration of the sodium/proton(Na⁺/H⁺) exchange transport system, for example, cardiovascular diseases(e.g., arteriosclerosis, hypertension, arrhythmia (e.g. ischemicarrhythmia, arrhythmia due to myocardial infarction, myocardialstunning, myocardial dysfunction, arrhythmia after percutaneoustransluminal coronary angioplasty (PTCA) or after thrombolysis, etc.),angina pectoris, cardiac hypertrophy, myocardial infarction, heartfailure (e.g., congestive heart failure, acute heart failure, cardiachypertrophy, etc.), restenosis after PTCA, PTCI, shock (e.g.,hemorrhagic shock, endotoxin shock, etc.)), renal diseases (e.g.,diabetes mellitus, diabetic nephropathy, ischemic acute renal failure,etc.) organ disorders associated with ischemia or ischemic reperfusion(e.g., heart muscle ischemic reperfusion associated disorders, acuterenal failure, or disorders induced by surgical treatment such ascoronary artery bypass grafting (CABG) surgeries, vascular surgeries,organ transplantation, non-cardiac surgeries or percutaneoustransluminal coronary angioplasty (PTCA)), cerebrovascular diseases(e.g., ischemic stroke, hemorrhagic stroke, etc.), cerebro ischemicdisorders (e.g., disorders associated with cerebral infarction,disorders caused after cerebral apoplexy as sequelae, or cerebraledema).

NHE-1 inhibitors are disclosed in U.S. Pat. No. 5,698,581; EuropeanPatent Application Publication No. EP 803 501 A1; and InternationalPatent Application Publication Nos. WO 94/26709; and WO 98/26803; eachof which is incorporated herein by reference. The NHE-1 inhibitorsdisclosed therein have utility in the combination aspects of the presentinvention. Said NHE-1 inhibitors can be prepared as disclosed therein.

Preferred NHE-1 inhibitors include compounds of the formula NHE,

prodrugs thereof or pharmaceutically acceptable salts of said compoundsand said prodrugs, wherein the variables are as defined in InternationalPatent Application Publication No. WO 99/43663, which is incorporatedherein by reference.

Especially preferred NHE-1 inhibitors include[1-(8-bromoquinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(6-chloroquinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(indazol-7-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(benzimidazol-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(1-isoquinolyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[5-cyclopropyl-1-(4-quinolinyl)-1H-pyrazole-4-carbonyl]guanidine;[5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine;[5-cyclopropyl-1-(quinolin-8-yl)-1H-pyrazole-4-carbonyl]guanidine;[1-(indazol-6-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(indazol-5-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(benzimidazol-5-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(1-methylbenzimidazol-6-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(5-quinolinyl)-5-n-propyl-1H-pyrazole-4-carbonyl]guanidine;[1-(5-quinolinyl)-5-isopropyl-1H-pyrazole-4-carbonyl]guanidine;[5-ethyl-1-(6-quinolinyl)-1H-pyrazole-4-carbonyl]guanidine;[1-(2-methylbenzimidazol-5-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(1,4-benzodioxan-6-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(benzotriazol-5-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(3-chloroindazol-5-yl)-5-ethyl-1H-pyrazole-4-carbonyl]guanidine;[1-(5-quinolinyl)-5-butyl-1H-pyrazole-4-carbonyl]guanidine;[5-propyl-1-(6-quinolinyl)-1H-pyrazole-4-carbonyl]guanidine;[5-isopropyl-1-(6-quinolinyl)-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chloro-4-methylsulfonylphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chlorophenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-trifluoromethyl-4-fluorophenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-bromophenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-fluorophenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chloro-5-methoxyphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chloro-4-methylaminosulfonylphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2,5-dichlorophenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2,3-dichlorophenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chloro-5-aminocarbonylphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chloro-5-aminosulfonylphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-fluoro-6-trifluoromethylphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chloro-5-methylsulfonylphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chloro-5-dimethylaminosulfonylphenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-trifluoromethyl-4-chlorophenyl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine;[1-(2-chlorophenyl)-5-methyl-1H-pyrazole-4-carbonyl]guanidine;[5-methyl-1-(2-trifluoromethylphenyl)-1H-pyrazole-4-carbonyl]guanidine;[5-ethyl-1-phenyl-1H-pyrazole-4-carbonyl]guanidine;[5-cyclopropyl-1-(2-trifluoromethylphenyl)-1H-pyrazole-4-carbonyl]guanidine;[5-cyclopropyl-1-phenyl-1H-pyrazole-4-carbonyl]guanidine;[5-cyclopropyl-1-(2,6-dichlorophenyl)-1H-pyrazole-4-carbonyl]guanidine;and pharmaceutically acceptable salts thereof.

The preferred and especially preferred NHE-1 inhibitors disclosed in theabove two paragraphs can be prepared according to methods set forth inInternational Patent Application Publication No. WO 99/43663, asreferenced above.

Preferably, the compounds of formula I of this invention can be used incombination with NHE-1 inhibitors as agents for myocardial protectionbefore, during, or after coronary artery bypass grafting (CABG)surgeries, vascular surgeries, percutaneous transluminal coronaryangioplasty (PTCA), organ transplantation or non-cardiac surgeries.Preferably, the compounds of formula I of this invention can be used incombination with NHE-1 inhibitors as agents for myocardial protection inpatients presenting with ongoing cardiac (acute coronary syndromes,e.g., myocardial infarction or unstable angina) or cerebral ischemicevents (e.g., stroke). Preferably, the compounds of formula I of thisinvention can be used in combination with NHE-1 inhibitors as agents forchronic myocardial protection in patients with diagnosed coronary heartdisease (e.g., previous myocardial infarction or unstable angina) orpatients who are at high risk for myocardial infarction (age greaterthan 65 and two or more risk factors for coronary heart disease).

In addition, a combination of the compounds of formula I of the presentinvention and NHE-1 inhibitors has a strong inhibitory effect on theproliferation of cells, for example the proliferation of fibroblastcells and the proliferation of the smooth muscle cells of the bloodvessels. For this reason, the combination of the compounds of formula Iof the present invention and NHE-1 inhibitors is a valuable therapeuticagent for use in diseases in which cell proliferation represents aprimary or secondary cause and may, therefore, be used asantiatherosclerotic agents, and as agents against diabetic latecomplications, cancerous diseases, fibrotic diseases such as pulmonaryfibrosis, hepatic fibrosis or renal fibrosis, glomerularnephrosclerosis, organ hypertrophies or hyperplasias, in particularhyperplasia or hypertrophy of the prostate, pulmonary fibrosis, diabeticcomplications or recurrent stricture after PTCA, or diseases caused byendothelial cell injury.

The utility of the combination of compounds of the present invention andNHE-1 inhibitors as medical agents in the treatment of diseases, such asare detailed herein, in mammals (e.g., humans), for example, myocardialprotection during surgery or myocardial protection in patientspresenting with ongoing cardiac or cerebral ischemic events or chroniccardioprotection in patients with diagnosed coronary heart disease, orat risk for coronary heart disease, cardiac dysfunction or myocardialstunning is demonstrated by the activity of said combination inconventional preclinical cardioprotection assays, as reported in thescientific literature and in WO 99/43663. The therapeutic effects of thecombination of the compounds of formula I of the present invention andNHE-1 inhibitors in preventing heart tissue damage resulting from anischemic insult can be demonstrated in vitro utilizing proceduresreported in the scientific literature and in WO 99/43663. Thetherapeutic effects of a combination of a compound of formula I of thepresent invention and an NHE-1 inhibitor in preventing heart tissuedamage otherwise resulting from an ischemic insult can also bedemonstrated in vivo utilizing procedures reported in the scientificliterature and in WO 99/43663.

The combination of a compound of formula I of the present invention andan NHE-1 inhibitor can be tested for their utility in reducing orpreventing ischemic injury in non-cardiac tissues, for example, thebrain, or the liver, utilizing procedures reported in the scientificliterature and in WO 99/43663. The combination of a compound of formulaI of the present invention and an NHE-1 inhibitor in such tests can beadministered by the preferred route and vehicle of administration and atthe preferred time of administration either prior to the ischemicepisode, during the ischemic episode, following the ischemic episode(reperfusion period) or during any of the experimental stages, asreferenced herein.

Compositions containing both a compound of formula I of the presentinvention and a NHE-1 inhibitor will generally be administered orally orparenterally at dosages between about 0.001 and 100 mg of said compoundof formula I of the present invention per kg body weight of the subjectto be treated per day and about 0.001 to 100 mg/kg/day of the NHE-1inhibitor. An especially preferred dosage contains between about 0.01and 10 mg/kg/day of said compound of formula I of the present inventionand between about 0.01 and 50 mg/kg/day of said NHE-1 inhibitor. Thecompositions of the present invention comprising a compound of formula Iof the present invention in combination with an NHE-1 inhibitor areuseful, for example, in reducing or minimizing damage effected directlyto any tissue that may be susceptible to ischemia/reperfusion injury(e.g., heart, brain, lung, kidney, liver, gut, skeletal muscle, retina)as the result of an ischemic event (e.g., myocardial infarction).Therefore, the composition is usefully employed prophylactically toprevent, i.e. (prospectively or prophylactically) to blunt or stemtissue damage (e.g., myocardial tissue) in patients who are at risk forischemia (e.g., myocardial ischemia).

Any glycogen phosphorylase inhibitor may be used as the Second Agent inthe combination compositions, combination therapies and kits of thepresent invention. The term glycogen phosphorylase inhibitor refers toany substance or agent or any combination of substances and/or agentswhich reduces, retards, or eliminates the enzymatic action of glycogenphosphorylase. The currently known enzymatic action of glycogenphosphorylase is the degradation of glycogen by catalysis of thereversible reaction of a glycogen macromolecule and inorganic phosphateto glucose-1-phosphate and a glycogen macromolecule which is oneglucosyl residue shorter than the original glycogen macromolecule(forward direction of glycogenolysis). Such actions are readilydetermined by those skilled in the art according to standard assaysknown in the art. A variety of these compounds are included in U.S. Pat.No. 5,988,463 and in the following published PCT patent applications: WO96/39384 and WO96/39385. However, other glycogen phosphorylaseinhibitors will be known to those skilled in the art.

Compositions containing both a compound of formula I and a glycogenphosphorylase inhibitor will generally be administered orally orparenterally at dosages between about 0.001 and 100 mg of said compoundof formula I of the present invention per kg body weight of the subjectto be treated per day and 0.005 to 50 mg/kg/day of said glycogenphosphorylase inhibitor, preferably 0.01 and 10 mg/kg/day of saidcompound of formula I of the present invention and 0.01 to 25 mg/kg/dayof said glycogen phosphorylase inhibitor, and most preferably 0.01 and10 mg/kg/day of said compound of formula I of the present invention and0.1 to 15 mg/kg/day of said glycogen phosphorylase inhibitor. However,some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

Any selective serotonin reuptake inhibitor (SSRI) may be used as theSecond Agent in the combination compositions, combination therapies andkits of the present invention. The term selective serotonin reuptakeinhibitor refers to an agent which inhibits the reuptake of serotonin byafferent neurons. Such inhibition is readily determined by those skilledin the art according to standard assays such as those disclosed in U.S.Pat. No. 4,536,518 and other U.S. patents recited in the next paragraph.

Preferred selective serotonin reuptake inhibitors which may be used inaccordance with the present invention include femoxetine, which may beprepared as described in U.S. Pat. No. 3,912,743; fluoxetine, which maybe prepared as described in U.S. Pat. No. 4,314,081; fluvoxamine, whichmay be prepared as described in U.S. Pat. No. 4,085,225; indalpine,which may be prepared as described in U.S. Pat. No. 4,064,255;indeloxazine, which may be prepared as described in U.S. Pat. No.4,109,088; milnacipran, which may be prepared as described in U.S. Pat.No. 4,478,836; paroxetine, which may be prepared as described in U.S.Pat. No. 3,912,743 or U.S. Pat. No. 4,007,196; sertraline and itspharmaceutically acceptable acid addition salts, such as thehydrochloride salt, which may be prepared as described in U.S. Pat. No.4,536,518; sibutramine, which may be prepared as described in U.S. Pat.No. 4,929,629; and zimeldine, which may be prepared as described in U.S.Pat. No. 3,928,369. Fluoxetine is also known as Prozac®. Sertralinehydrochloride is also known as Zoloft®. Sibutramine is also known asMeridia®. The disclosures thereof are incorporated herein by reference.

Selective serotonin reuptake inhibitors are preferably administered inamounts ranging from about 0.01 mg/kg/day to about 500 mg/kg/day insingle or divided doses, preferably about 10 mg to about 300 mg per dayfor an average subject, depending upon the selective serotonin reuptakeinhibitor and the route of administration. However, some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The individual responsible for dosing will, in any event,determine the appropriate dose for the individual subject.

Any 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitormay be used as the Second Agent in the combination compositions,combination therapies and kits of the present invention. The term3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitorrefers to a pharmaceutical agent which inhibits the enzyme3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzymeis involved in the conversion of HMG-CoA to mevalonate, which is one ofthe steps in cholesterol biosynthesis. Such inhibition is readilydetermined according to standard assays well known to those skilled inthe art.

Preferred 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitorswhich may be used in accordance with the present invention includeatorvastatin, disclosed in U.S. Pat. No. 4,681,893, atorvastatincalcium, disclosed in U.S. Pat. No. 5,273,995, cerivastatin, disclosedin U.S. Pat. No. 5,502,199, dalvastatin, disclosed in European PatentApplication Publication No. 738,510 A2, fluindostatin, disclosed inEuropean Patent Application Publication No. 363,934 A1, fluvastatin,disclosed in U.S. Pat. No. 4,739,073, lovastatin, disclosed in U.S. Pat.No. 4,231,938, mevastatin, disclosed in U.S. Pat. No. 3,983,140,pravastatin, disclosed in U.S. Pat. No. 4,346,227, simvastatin,disclosed in U.S. Pat. No. 4,444,784 and velostatin, disclosed in U.S.Pat. No. 4,448,784 and U.S. Pat. No. 4,450,171, all of which areincorporated herein by reference. Especially preferred3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors includeatorvastatin, atorvastatin calcium, also known as Lipitor®, lovastatin,also known as Mevacor®, pravastatin, also known as Pravachol®, andsimvastatin, also known as Zocor®.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors arepreferably administered in amounts ranging from about 0.1 mg/kg to about1000 mg/kg/day in single or divided doses, preferably about 1 mg/kg/dayto about 200 mg/kg/day for an average subject, depending upon the3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor and the routeof administration. However, some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theindividual responsible for dosing will, in any event, determine theappropriate dose for the individual subject.

Any thiazolidinedione antidiabetic agent may be used in the combinationcompositions, combination therapies and kits of the present invention.The term thiazolidinedione antidiabetic agent refers to a pharmaceuticalagent that increases insulin sensitivity in tissues important forinsulin action such as adipose tissue, skeletal muscle, and liver.

The following patents exemplify thiazolidinedione antidiabetic agentswhich can be used in the combination compositions, methods and kits ofthe present invention: U.S. Pat. No. 4,340,605; U.S. Pat. No. 4,342,771;U.S. Pat. No. 4,367,234; U.S. Pat. No. 4,617,312; U.S. Pat. No.4,687,777 and U.S. Pat. No. 4,703,052. Preferred thiazolidinedioneantidiabetic agents include pioglitazone, also known as Actos®, androsiglitazone, also known as Avandia®.

Thiazolidinedione antidiabetic agents are preferably administered inamounts ranging from about 0.1 mg/day to about 100 mg/day in single ordivided doses, preferably about 0.1 mg/day to about 50 mg/day for anaverage subject, depending upon the thiazolidinedione antidiabetic agentand the route of administration. However, some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The individual responsible for dosing will, in any event,determine the appropriate dose for the individual subject.

Any angiotensin converting enzyme (ACE) inhibitor may be used as theSecond Agent in the combination compositions, combination therapies andkits of the present invention. The term angiotensin converting enzymeinhibitor refers to a pharmaceutical agent which inhibits angiotensinconverting enzyme activity. Angiotensin converting enzyme is involved inthe conversion of angiotensin I to the vasoconstrictor, angiotensin II.The activity of angiotensin converting enzyme inhibitors may readily bedetermined by methods known to those skilled in the art, including anyof the standard assays described in the patents listed below.

Preferred angiotensin converting enzyme inhibitors include: alacepril,disclosed in U.S. Pat. No. 4,248,883; benazepril, disclosed in U.S. Pat.No. 4,410,520; captopril, disclosed in U.S. Pat. Nos. 4,046,889 and4,105,776; ceronapril, disclosed in U.S. Pat. No. 4,452,790; delapril,disclosed in U.S. Pat. No. 4,385,051; enalapril, disclosed in U.S. Pat.No. 4,374,829; fosinopril, disclosed in U.S. Pat. No. 4,337,201;imadapril, disclosed in U.S. Pat. No. 4,508,727; lisinopril, disclosedin U.S. Pat. No. 4,555,502; moexipril, disclosed in U.S. Pat. No.4,344,949; moveltopril, disclosed in Belgian Patent No. 893,553;perindopril, disclosed in U.S. Pat. No. 4,508,729; quinapril and itshydrochloride salt, disclosed in U.S. Pat. No. 4,344,949; ramipril,disclosed in U.S. Pat. No. 4,587,258; spirapril, disclosed in U.S. Pat.No. 4,470,972; temocapril, disclosed in U.S. Pat. No. 4,699,905; andtrandolapril, disclosed in U.S. Pat. No. 4,933,361. The disclosures ofall such patents are incorporated herein by reference.

Angiotensin converting enzyme inhibitors are preferably administered inamounts ranging from about 0.01 mg/kg/day to about 500 mg/kg/day insingle or divided doses, preferably about 10 mg to about 300 mg per dayfor an average subject, depending upon the angiotensin converting enzymeinhibitor and the route of administration. However, some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The individual responsible for dosing will, in any event,determine the appropriate dose for the individual subject.

Any angiotensin-II receptor (A-II) antagonist may be used as the SecondAgent in the combination compositions, combination therapies and kits ofthe present invention. The term angiotensin-II receptor antagonistrefers to a pharmaceutical agent that blocks the vasoconstrictor effectsof angiotensin II by blocking the binding of angiotensin II to the AT₁receptor found in many tissues, (e.g., vascular smooth muscle, adrenalgland). The activity of angiotensin-II receptor antagonist may readilybe determined by methods known to those skilled in the art, includingany of the standard assays described in the patents listed below.

Angiotensin-II receptor antagonists include: candesartan, which may beprepared as disclosed in U.S. Pat. No. 5,196,444; eprosartan, which maybe prepared as disclosed in U.S. Pat. No. 5,185,351; irbesartan, whichmay be prepared as disclosed in U.S. Pat. No. 5,270,317; losartan, whichmay be prepared as disclosed in U.S. Pat. No. 5,138,069; and valsartan,which may be prepared as disclosed in U.S. Pat. No. 5,399,578. Thedisclosures thereof are incorporated herein by reference. More preferredangiotensin-II receptor antagonists are losartan, irbesartan andvalsartan.

Angiotensin-II receptor antagonists are preferably administered inamounts ranging from about 0.01 mg/kg/day to about 500 mg/kg/day insingle or divided doses, preferably about 10 mg to about 300 mg per dayfor an average subject, depending upon the angiotensin-II receptorantagonist and the route of administration. However, some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The individual responsible for dosing will, in any event,determine the appropriate dose for the individual subject.

Any γ-aminobutyric acid (GABA) agonist may be used as the Second Agentin the combination compositions, combination therapies and kits of thepresent invention. The term γ-aminobutyric acid agonist refers to apharmaceutical agent that binds to GABA receptors in the mammaliancentral nervous system. GABA is the major inhibitory neurotransmitter inthe mammalian central nervous system. The activity of γ-aminobutyricacid (GABA) agonist may readily be determined by methods known to thoseskilled in the art, including the procedures disclosed in Janssens deVerebeke, P. et al., Biochem. Pharmacol., 31, 2257-2261 (1982), Loscher,W., Biochem. Pharmacol., 31, 837-842, (1982) and/or Phillips, N. et al.,Biochem. Pharmacol., 31, 2257-2261.

Preferred γ-aminobutyric acid agonists, which may be prepared byprocedures available in the art, include: muscimol, progabide, riluzole,baclofen, gabapentin (Neurontin®), vigabatrin, valproic acid, tiagabine(Gabitril®), lamotrigine (Lamictal®), pregabalin, pagoclone, phenytoin(Dilantin®), carbamazepine (Tegretol®), topiramate (Topamax®) andanalogs, derivatives, prodrugs and pharmaceutically acceptable salts ofthose γ-aminobutyric acid agonist agonists.

In general, in accordance with the present invention, the γ-aminobutyricacid agonist used in the combinations, pharmaceutical compositions,methods and kits of the present invention will be administered in adosage amount of about 4 mg/kg body weight of the subject to be treatedper day to about 60 mg/kg body weight of the subject to be treated perday, in single or divided doses. However, some variation in dosage willnecessarily occur depending upon the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Inparticular, when used as the γ-aminobutyric acid agonist agonist in thepresent invention, pregabalin will be dosed at about 300 mg to about1200 mg per day; gabapentin will be dosed at about 600 mg to about 3600mg per day.

Any phosphodiesterase type 5 (PDE-5) inhibitor may be used as the SecondAgent in the combination compositions, combination therapies and kits ofthe present invention. The term phosphodiesterase type 5 inhibitorrefers to any substance or agent or any combination of substances and/oragents which reduces, retards, or eliminates the enzymatic action ofcyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5.Such actions are readily determined by those skilled in the artaccording to assays as described in PCT application publication WO00/24745.

The following patent publications exemplify phosphodiesterase type 5inhibitors which can be used in the combination compositions, methodsand kits of the present invention, and refer to methods of preparingthose phosphodiesterase type 5 (PDE-5) inhibitors: PCT applicationpublication WO 00/24745; PCT application publication WO 94/28902;European Patent application publication 0463756A1; European Patentapplication publication 0526004A1 and European Patent applicationpublication 0201188A2. A preferred phosphodiesterase type 5 inhibitor issildenafil citrate, also known as VIAGRA®.

Suitable cGMP PDE5 inhibitors for the use according to the presentinvention include: the pyrazolo [4,3-d]pyrimidin-7-ones disclosed inEP-A-0463756; the pyrazolo [4,3-d]pyrimidin-7-ones disclosed inEP-A-0526004; the pyrazolo [4,3-d pyrimidin-7-ones disclosed inpublished international patent application WO 93/06104; the isomericpyrazolo [3,4-d]pyrimidin-4-ones disclosed in published internationalpatent application WO 93/07149; the quinazolin-4-ones disclosed inpublished international patent application WO 93/12095; the pyrido[3,2-d]pyrimidin-4-ones disclosed in published international patentapplication WO 94/05661; the purin-6-ones disclosed in publishedinternational patent application WO 94/00453; the pyrazolo[4,3-d]pyrimidin-7-ones disclosed in published international patentapplication WO 98/49166; the pyrazolo [4,3-d]pyrimidin-7-ones disclosedin published international patent application WO 99/54333; the pyrazolo[4,3-d]pyrimidin-4-ones disclosed in EP-A-0995751; the pyrazolo[4,3-d]pyrimidin-7-ones disclosed in published international patentapplication WO 00/24745; the pyrazolo [4,3-d]pyrimidin-4-ones disclosedin EP-A-0995750; the compounds disclosed in published internationalapplication WO95/19978; the compounds disclosed in publishedinternational application WO 99/24433; the compounds disclosed inpublished international application WO 93/07124; the compounds disclosedin international patent application PCT IB 00/01457 filed on 11^(th)Oct. 2000 and the compounds disclosed in international patentapplication PCT IB 00/01430 filed on 4^(th) Oct. 2000. It is to beunderstood that the contents of the above published patent applications,and in particular the general formulae and exemplified compounds thereinare incorporated herein in their entirety by reference thereto.

Preferred type V phosphodiesterase inhibitors for the use according tothe present invention include:5-[2-ethoxy-5-(4-methyl-1-piperazinylsulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(sildenafil) also known as1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulphonyl]-4-methylpiperazine(see EP-A-0463756);5-(2-ethoxy-5-morpholinoacetylphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see EP-A-0526004);3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-n-propoxyphenyl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see WO98/49166);3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxyethoxy)pyridin-3-yl]-2-(pyridin-2-yl)methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see WO99/54333);(+)-3-ethyl-5-[5-(4-ethylpiperazin-1-ylsulphonyl)-2-(2-methoxy-1(R)-methylethoxy)pyridin-3-yl]-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,also known as3-ethyl-5-{5-[4-ethylpiperazin-1-ylsulphonyl]-2-([(1R)-2-methoxy-1-methylethyl]oxy)pyridin-3-yl}-2-methyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see WO99/54333);5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,also known as1-{6-ethoxy-5-[3-ethyl-6,7-dihydro-2-(2-methoxyethyl)-7-oxo-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-pyridylsulphonyl}-4-ethylpiperazine(see PCT IB 00/01457);5-[2-iso-Butoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-(1-methylpiperidin-4-yl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see PCT IB 00/01457);5-[2-Ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-phenyl-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see PCT IB 00/01457);5-(5-Acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see PCT IB 00/01430);5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(see PCT IB 00/01430);(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione(IC-351), i.e. the compound of examples 78 and 95 of publishedinternational application WO95/19978, as well as the compound ofexamples 1, 3, 7 and 8;2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one(vardenafil) also known as1-[[3-(3,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-f]-as-triazin-2-yl)-4-ethoxyphenyl]sulphonyl]-4-ethylpiperazine,i.e. the compound of examples 20, 19, 337 and 336 of publishedinternational application WO99/24433; and the compound of example 11 ofpublished international application WO93/07124 (EISAI); and compounds 3and 14 from Rotella D P, J. Med. Chem., 2000, 43, 1257.

Still other type cGMP PDE5 inhibitors useful in conjunction with thepresent inventioninclude:4-bromo-5-(pyridylmethylamino)-6-[3-(4-chlorophenyl)-propoxy]-3(2H)pyridazinone;1-[4-[(1,3-benzodioxol-5-ylmethyl)amiono]-6-chloro-2-quinozolinyl]-4-piperidine-carboxylicacid, monosodium salt;(+)-cis-5,6a,7,9,9,9a-hexahydro-2-[4-(trifluoromethyl)-phenylmethyl-5-methyl-cyclopent-4,5]imidazo[2,1-b]purin-4(3H)one;furazlocillin;cis-2-hexyl-5-methyl-3,4,5,6a,7,8,9,9a-octahydrocyclopent[4,5]-imidazo[2,1-b]purin-4-one;3-acetyl-1-(2-chlorobenzyl)-2-propylindole-6-carboxylate;3-acetyl-1-(2-chlorobenzyl)-2-propylindole-6-carboxylate;4-bromo-5-(3-pyridylmethylamino)-6-(3-(4-chlorophenyl)propoxy)-3-(2H)pyridazinone;1-methyl-5(5-morpholinoacetyl-2-n-propoxyphenyl)-3-n-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one;1-[4-[(1,3-benzodioxol-5-ylmethyl)amino]-6-chloro-2-quinazolinyl]-4-piperidinecarboxylicacid, monosodium salt; Pharmaprojects No. 4516 (Glaxo Wellcome);Pharmaprojects No. 5051 (Bayer); Pharmaprojects No. 5064 (Kyowa Hakko;see WO 96/26940); Pharmaprojects No. 5069 (Schering Plough); GF-196960(Glaxo Wellcome); E-8010 and E-4010 (Eisai); Bay-38-3045 & 38-9456(Bayer) and Sch-51866.

Highly preferred herein are sildenafil, IC-351, vardenafil,5-(5-Acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-oneand5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one.

The suitability of any particular cGMP PDE5 inhibitor can be readilydetermined by evaluation of its potency and selectivity using literaturemethods followed by evaluation of its toxicity, absorption, metabolism,pharmacokinetics, etc in accordance with standard pharmaceuticalpractice. Preferably, the cGMP PDE5 inhibitors have an IC50 at less than100 nanomolar, more preferably, at less than 50 nanomolar, morepreferably still at less than 10 nanomolar. IC50 values for the cGMPPDE5 inhibitors may be determined using established literaturemethodology, for example as described in EP0463756-B1 and EP0526004-A1.Preferably the cGMP PDE5 inhibitors used in the invention are selectivefor the PDE5 enzyme. Preferably they are selective over PDE3, morepreferably over PDE3 and PDE4. Preferably, the cGMP PDE5 inhibitors ofthe invention have a selectivity ratio greater than 100 more preferablygreater than 300, over PDE3 and more preferably over PDE3 and PDE4.Selectivity ratios may readily be determined by the skilled person. IC50values for the PDE3 and PDE4 enzyme may be determined using establishedliterature methodology, see S A Ballard et al, Journal of Urology, 1998,vol. 159, pages 2164-2171.

Phosphodiesterase type 5 inhibitors are preferably administered inamounts ranging from about 5 mg/day to about 500 mg/day in single ordivided doses, preferably about 10 mg/day to about 250 mg/day, for anaverage subject depending upon the phosphodiesterase type 5 inhibitorand the route of administration. However, some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The individual responsible for dosing will, in any event,determine the appropriate dose for the individual subject.

Any adenosine agonist may be used as the Second Agent in the combinationcompositions, combination therapies and kits of the present invention.The term adenosine agonist refers to any substances and/or agents whichpharmacologically affect the cardioprotective effects of ischemicpreconditioning by activating adenosine A-3 receptors.

The utility of the adenosine agonists as medical agents in the treatmentof cardiac tissue ischemia is demonstrated by the activity of saidagonists in conventional preclinical cardioprotection assays (see the invivo assay in Klein, H. et al., Circulation 92:912-917 (1995); theisolated heart assay in Tracey, W. R. et al., Cardiovascular Research33:410-415 (1997); the antiarrhythmic assay in Yasutake M. et al., Am.J. Physiol., 36:H2430-H2440 (1994); the NMR assay in Kolke et al., J.Thorac. Cardiovasc. Surg. 112: 765-775 (1996)) and the additional invitro and in vivo assays described below. Such assays also provide ameans whereby the activities of adenosine agonists can be compared withthe activities of other known compounds. The results of thesecomparisons are useful for determining dosage levels in mammals,including humans, for the treatment of such diseases.

Human Adenosine A1 and A3 Receptor Assays

Materials: Full-length human adenosine A₁ and A₃ receptor cDNA'ssubcloned into the eukaryotic expression vector pRcCMV (Invitrogen) werepurchased from The Garvan Institute, Sydney, Australia. Chinese hamsterovary (CHO-K1) cells were obtained from the American Type Tissue CultureCollection (Rockville, Md., USA). DMEM and DMEM/F12 culture media andfoetal calf serum were obtained from Gibco-BRL (Grand Island, N.Y.,USA). The A1/A3 adenosine receptor agonistN6-(4-amino-3-[125I]iodobenzyl)adenosine (¹²⁵I-ABA) was prepared by NewEngland Nuclear (Boston, Mass., USA). Adenosine deaminase (ADA) wasobtained from Boehringer Mannheim (Indianapolis, Ind., USA). Thephosphodiesterase inhibitor RO-20-1724 was obtained from ResearchBiochemicals International (Natick, Mass., USA).

Expression of Human Adenosine A1 and A3 Receptors

For stable expression studies, adenosine receptor A₁ and A₃ expressionplasmids (20 μg) are transfected into CHO-K1 cells, or HEK 293s cells,respectively, grown in DMEM/F12 (CHO) or DMEM (HEK 293s), with 10%foetal calf serum media, using a calcium phosphate mammalian celltransfection kit (5 Prime-3 Prime). Stable transfectants are obtained byselection in complete media containing 500 μg/ml (CHO) or 700 μg/ml (HEK293s) active neomycin (G418) and screened for expression by [¹²⁵I]-ABAbinding.

Receptor Membrane Preparation

Cells stably expressing either human A₁ or human A₃ receptors arecollected by centrifugation at 300×g for 5 minutes, the supernatant isdiscarded and the cell pellet is resuspended in cell buffer consistingof (mmoles/L): HEPES (10), MgCl₂ (5), PMSF (0.1), bacitracin (100μg/ml), leupeptin (10 μg/ml), DNAse I (100 μg/ml), ADA (2 U/ml), pH 7.4.Crude cell membranes are prepared by repeated aspiration through a 21gauge needle, collected by centrifugation at 60,000×g for 10 minutes andstored in cell buffer at −80° C.

Estimation of Compound Binding Affinity Constants (K_(i))

Receptor membranes are resuspended in incubation buffer consisting of(mmoles/L): HEPES (10), EDTA (1), MgCl₂ (5), pH 7.4. Binding reactions(10-20 μg membrane protein) are carried out for one hour at roomtemperature in 250 μl incubation buffer containing 0.1 nM of ¹²⁵I-ABA(2200 Ci/mmol) and increasing concentrations of compound (0.1 nM-30 μM).The reaction is stopped by rapid filtration with ice-cold PBS, throughglass fibre filters (presoaked in 0.6% polyethylenimine) using a Tomtec96-well harvester (Orange, Conn., USA). Filters are counted in a WallacMicrobeta liquid scintillation counter (Gaithersberg, Md., USA).Nonspecific binding is determined in the presence of 5 μM I-ABA.Compound inhibitory constants (K_(i)) are calculated by fitting bindingdata via nonlinear least squares regression analysis to the equation: %Inhibition=100/[1+(10^(C)/10^(X))^(D)], where X=log[compoundconcentration], C (IC₅₀)=log[compound concentration at 50% inhibtion],and D=the Hill slope. At the concentration of radioligand used in thepresent study (10 fold<K_(D)), IC₅₀=K_(i).

Assessment of Human Adenosine A3 Receptor Agonist Activity

Adenosine A3 agonist activity is assessed by compound inhibition ofisoproterenol-stimulated cAMP levels. HEK293s cells stably transfectedwith human A3 receptors (as described above) are washed with PhosphateBuffered Saline (PBS) (Ca/Mg-free) and detached with 1.0 mM EDTA/PBS.Cells are collected by centrifugation at 300×g for 5 minutes and thesupernatant discarded. The cell pellet is dispersed and resuspended incell buffer (DMEM/F12 containing 10 mM HEPES, 20 μM RO-20-1724 and 1U/ml ADA). Following preincubation of cells (100,000/well) for 10 min at37° C., 1 μM isoproterenol, with or without increasing concentrations(0.1 nM-300 nM) test compound, and the incubation is continued for 10min. Reactions are terminated by the addition of 1.0 N HCl followed bycentrifugation at 2000×g for 10 minutes. Sample supernatants (10 μl) areremoved and cAMP levels determined by radioimmunoassay (New EnglandNuclear, Boston, Mass., USA). The basal and controlisoproterenol-stimulated cAMP accumulation (pmol/ml/100,000 cells) areroutinely 3 and 80, respectively. Smooth curves are fitted to the datavia nonlinear least squares regression analysis to the equation: %isoproterenol-stimulated cAMP=100/[1+(10^(X)/10^(C))^(D)], whereX=log[compound concentration], C (IC₅₀)=log[compound concentration at50% inhibition], and D=the Hill slope.

The results from an in vitro test, such as that described in Tracey etal., Cardiovasc. Res., 33:410-415, 1997, demonstrate that adenosineagonists induce significant cardioprotection relative to the controlgroup. The following patent publications exemplify adenosine agonistswhich can be used in the combination compositions, methods and kits ofthe present invention, and refer to methods of preparing those adenosineagonists: U.S. Pat. No. 5,604,210; U.S. Pat. No. 5,688,774; U.S. Pat.No. 5,773,423; J. Med. Chem. 1994, 37, 636-646; J. Med. Chem. 1995, 38,1174-1188; J. Med. Chem. 1995, 38, 1720-1735.

U.S. Pat. No. 5,817,760 discloses recombinant human adenosine receptorsA1, A2a, A2b, and A3 which were prepared by cDNA cloning and polymerasechain reaction techniques. The recombinant adenosine receptors can beutilized in an assay to identify and evaluate entities that bind to orenhance binding to adenosine receptors.

Adenosine agonists are preferably administered in amounts ranging fromabout 0.001 mg/kg/day to about 100 mg/kg/day, for an average subjectdepending upon the adenosine agonist and the route of administration. Anespecially preferred dosage is about 0.01 mg/kg/day to about 50mg/kg/day of an adenosine agonists. However, some variation in dosagewill necessarily occur depending upon the condition of the subject beingtreated. The individual responsible for dosing will, in any event,determine the appropriate dose for the individual subject.

Any compound having activity as a CETP inhibitor can serve as the SecondAgent in the combination compositions, combination therapies and kits ofthe present invention. The term CETP inhibitor refers to compounds thatinhibit the cholesteryl ester transfer protein (CETP) mediated transportof various cholesteryl esters and triglycerides from HDL to LDL andVLDL. A variety of these compounds are described and referenced below,however other CETP inhibitors will be known to those skilled in the art.U.S. Pat. No. 5,512,548 discloses certain polypeptide derivatives havingactivity as CETP inhibitors, while certain CETP-inhibitoryrosenonolactone derivatives and phosphate-containing analogs ofcholesteryl ester are disclosed in J. Antibiot., 49(8): 815-816 (1996),and Bioorg. Med. Chem. Lett.; 6:1951-1954 (1996), respectively. OtherCETP inhibitors that can be used in combination with compounds of thepresent invention are disclosed in WO 00/17164, WO 00/17165, WO99/20302, EP 796846, EP818197, EP 818448, WO 99/14204, WO 99/41237, WO95/04755, WO 96/15141, WO 96/05227, DE 19704244, DE19741051, DE19741399, DE 19704243, DE 19709125, DE 19627430, DE 19832159, DE19741400, JP 11049743, and JP 09059155. Preferred CETP inhibitors thatcan be used in combination with the compounds of the present inventioninclude

-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-methoxymethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid 2-hydroxy-ethyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-cyclopropyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid ethyl ester;-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid ethyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-cyclopropyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid propyl ester; and-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid propyl ester,-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-isopropyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-6-chloro-2-cyclopropyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2S,4S]2-cyclopropyl-4-[(3,5-dichloro-benzyl)-methoxycarbonyl-amino]-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-cyclopropyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid tert-butyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-cyclopropyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-cyclobutyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-methoxymethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid 2-hydroxy-ethyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-cyclopropyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid ethyl ester;-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid ethyl ester;-   [2S,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-cyclopropyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid propyl ester;-   [2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid propyl ester; and-   [2R,4S]4-[acetyl-(3,5-bis-trifluoromethyl-benzyl)-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylic    acid isopropyl ester;    and pharmaceutically acceptable salts and prodrugs thereof and salts    of the prodrugs.

In the aspects of this invention related to therapeutic methods oftreating or preventing diabetic complications wherein a compound offormula I of this invention and a Second Agent are administered togetheras part of the same pharmaceutical composition and to methods whereinthese two agents are administered separately, the appropriate dosageregimen, the amount of each dose administered and the intervals betweendoses of the active agents will again depend upon the compound offormula I of this invention and the Second Agent being used, the type ofpharmaceutical compositions being used, the characteristics of thesubject being treated and the severity of the condition, disease,symptom or complication being treated.

The compounds of the present invention may be administered alone or incombination with pharmaceutically acceptable vehicles, diluents orcarriers, in either single or multiple doses. Suitable pharmaceuticalcarriers include inert solid diluents or fillers, sterile aqueoussolutions and various organic solvents. The pharmaceutical compositionsformed by combining the compounds of formula I of the present inventionand the pharmaceutically acceptable carriers, vehicles or diluents arethen readily administered in a variety of dosage forms such as tablets,powders, lozenges, syrups, injectable solutions and the like. Thesepharmaceutical compositions can, if desired, contain additionalingredients such as flavorings, binders, excipients and the like. Thus,for purposes of oral administration, tablets containing variousexcipients such as sodium citrate, calcium carbonate and calciumphosphate may be employed along with various disintegrants such asstarch, alginic acid and certain complex silicates, together withbinding agents such as polyinylpyrrolidone, sucrose, gelatin and acacia.Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate or talc are often useful for tabletting purposes. Solidcompositions of a similar type may also be employed as fillers in softand hard filled gelatin capsules. Preferred materials for this includelactose or milk sugar and high molecular weight polyethylene glycols.When aqueous suspensions or elixirs are desired for oral administration,the essential active ingredient therein may be combined with varioussweetening or flavoring agents, coloring matter or dyes and, if desired,emulsifying or suspending agents, together with diluents such as water,ethanol, propylene glycol, glycerin or combinations thereof.

For parenteral administration, solutions of the compounds of the presentinvention in sesame or peanut oil, aqueous propylene glycol, or insterile aqueous solutions may be employed. Such aqueous solutions shouldbe suitably buffered if necessary and the liquid diluent first renderedisotonic with sufficient saline or glucose. These particular aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal administration. In this connection, thesterile aqueous media employed are all readily available by standardtechniques known to those skilled in the art.

Administration of the compounds of, formula I of the present inventioncan be via any method which delivers a compound of the present inventionpreferentially to the desired tissue (e.g., nerve, kidney, retina and/orcardiac tissues). These methods include oral, parenteral, topical,intraduodenal, rectal, inhalation routes, etc. Generally, the compoundsof the present invention are administered in single (e.g., once daily)or multiple doses or via constant infusion.

Generally, a compound of formula I of the present invention isadministered orally, or parenterally (e.g., intravenous, intramuscular,subcutaneous or intramedullary). Topical administration may also beindicated, for example, where the patient is suffering from agastrointestinal disorder or whenever the medication is best applied tothe surface of a tissue or organ as determined by the attendingphysician.

The amount and timing of compounds administered will, of course, bedependent on the subject being treated, on the severity of theaffliction, on the manner of administration and on the judgment of theprescribing physician. Thus, because of patient to patient variability,the dosages given below are a guideline and the physician may titratedoses of the drug to achieve the treatment that the physician considersappropriate for the patient. In considering the degree of treatmentdesired, the physician must balance a variety of factors such as age ofthe patient, presence of preexisting disease, as well as presence ofother diseases.

Thus, for example, in one mode of administration the compounds offormula I of the present invention may be administered just prior tosurgery (e.g., within twenty-four hours before surgery, for example,cardiac surgery) during or subsequent to surgery (e.g., withintwenty-four hours after surgery) where there is risk of myocardialischemia. The compounds of formula I of the present invention may alsobe administered in a chronic daily mode.

The compounds of the present invention are generally administered in theform of a pharmaceutical composition comprising at least one of thecompounds of formula I of this invention together with apharmaceutically acceptable vehicle or diluent. Thus, the compounds offormula I of this invention can be administered individually or togetherin any conventional oral, parenteral, rectal or transdermal dosage form.

For purposes of transdermal (e.g., topical) administration, dilutesterile, aqueous or partially aqueous solutions (usually in about 0.1%to 5% concentration), otherwise similar to the above parenteralsolutions, are prepared.

Methods of preparing various pharmaceutical compositions with a certainamount of an active ingredient, i.e, a compound of formula I of thepresent invention, are known, or will be apparent in light of thisdisclosure, to those skilled in this art. For examples of methods ofpreparing pharmaceutical compositions, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995).

Pharmaceutical compositions according to the present invention maycontain, for example, 0.0001%-95% of the compound(s) of the presentinvention. In any event, the composition or formulation to beadministered will contain a quantity of a compound(s) according to thepresent invention in an amount effective to treat thedisease/condition/complication of the subject being treated.

In the combination aspect of the present invention, a compound offormula I of the present invention and a Second Agent, as describedabove, can be co-administered simultaneously or sequentially in anyorder, or as a single pharmaceutical composition comprising a compoundof formula I of the present invention and a Second Agent.

Since the present invention has an aspect that relates to the treatmentof the disease/conditions/complications described herein with acombination of active ingredients which may be administered separately,the invention also relates to combining separate pharmaceuticalcompositions in kit form. The kit comprises two separate pharmaceuticalcompositions: a compound of formula I, a prodrug thereof or a salt ofsuch compound or prodrug, and a Second Agent as described above. The kitcomprises a container for containing the separate compositions such as adivided bottle or a divided foil packet. Typically the kit comprisesdirections for the administration of the separate components. The kitform is particularly advantageous when the separate components arepreferably administered in different dosage forms (e.g., oral andparenteral), are administered at different dosage intervals, or whentitration of the individual components of the combination is desired bythe prescribing physician.

An example of such a kit is a so-called blister pack. Blister packs arewell known in the packaging industry and are being widely used for thepackaging of pharmaceutical unit dosage forms (tablets, capsules, andthe like). Blister packs generally consist of a sheet of relativelystiff material covered with a foil of a preferably transparent plasticmaterial. During the packaging process recesses are formed in theplastic foil. The recesses have the size and shape of the tablets orcapsules to be packed. Next, the tablets or capsules are placed in therecesses and the sheet of relatively stiff material is sealed againstthe plastic foil at the face of the foil which is opposite from thedirection in which the recesses were formed. As a result, the tablets orcapsules are sealed in the recesses between the plastic foil and thesheet. Preferably the strength of the sheet is such that the tablets orcapsules can be removed from the blister pack by manually applyingpressure on the recesses whereby an opening is formed in the sheet atthe place of the recess. The tablet or capsule can then be removed viasaid opening.

It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows: “First Week, Monday,Tuesday, . . . etc . . . Second Week, Monday, Tuesday, . . . ” etc.Other variations of memory aids will be readily apparent. A “daily dose”can be a single tablet or capsule or several tablets or capsules to betaken on a given day. Also, a daily dose of a compound of formula I ofthe present invention can consist of one tablet or capsule while a dailydose of the Second Agent can consist of several tablets or capsules orvice versa. The memory aid should reflect this.

In another specific embodiment of the invention, a dispenser designed todispense the daily doses one at a time in the order of their intendeduse is provided. Preferably, the dispenser is equipped with amemory-aid, so as to further facilitate compliance with the regimen. Anexample of such a memory-aid is a mechanical counter which indicates thenumber of daily doses that has been dispensed. Another example of such amemory-aid is a battery-powered micro-chip memory coupled with a liquidcrystal readout, or audible reminder signal which, for example, readsout the date that the last daily dose has been taken and/or reminds onewhen the next dose is to be taken.

The compounds of formula I of the present invention generally will beadministered in a convenient formulation. The following formulationexamples are illustrative only and are not intended to limit the scopeof the present invention.

In the formulations which follow, “active ingredient” means acompound(s) of the present invention.

Formulation 1: Gelatin Capsules Hard gelatin capsules are prepared usingthe following: Ingredient Quantity (mg/capsule) Active ingredient0.25-100 Starch, NF   0-650 Starch flowable powder   0-50 Silicone fluid350 centistokes   0-15

A tablet formulation is prepared using the ingredients below:

Formulation 2: Tablets Ingredient Quantity (mg/tablet) Active ingredient0.25-100 Cellulose, microcrystalline  200-650 Silicon dioxide, fumed  10-650 Stearate acid   5-15

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.25-100 mg of active ingredientsare made up as follows:

Formulation 3: Tablets Ingredient Quantity (mg/tablet) Active ingredient0.25-100 Starch 45 Cellulose, microcrystalline 35 Polyvinylpyrrolidone(as 10% solution in water) 4 Sodium carboxymethyl cellulose 4.5Magnesium stearate 0.5 Talc 1

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.25-100 mg of active ingredient per 5 mldose are made as follows:

Formulation 4: Suspensions Ingredient Quantity (mg/5 ml) Activeingredient 0.25-100 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25mg Benzoic acid solution 0.10 mL Flavor q.v. Color q.v. Purified Waterto 5 mL

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form smoothpaste. The benzoic acid solution, flavor, and color are diluted withsome of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

An aerosol solution is prepared containing the following ingredients:

Formulation 5: Aerosol Ingredient Quantity (% by weight) Activeingredient 0.25 Ethanol 25.75 Propellant 22 (Chlorodifluoromethane)74.00

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

Formulation 6: Suppositories Ingredient Quantity (mg/suppository) Activeingredient 250 Saturated fatty acid glycerides 2,000

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

Formulation 7: Intravenous Solution Ingredient Quantity Activeingredient 25 mg-10,000 mg Isotonic saline 1,000 mL

The solution of the above ingredients is intravenously administered to apatient.

The active ingredient in the above formulations may also be acombination of active compounds.

General Experimental Procedures

Melting points were determined on a Thomas-Hoover capillary meltingpoint apparatus, and are uncorrected. ¹H NMR spectra were obtained on aBruker AM-250 (Bruker Co., Billerica, Mass.), a Bruker AM-300, a VarianXL-300 (Varian Co., Palo Alto, Calif.), or a Varian Unity 400 at about23° C. at 250, 300, or 400 MHz for proton. Chemical shifts are reportedin parts per million (δ) relative to residual chloroform (7.26 ppm),dimethylsulfoxide (2.49 ppm), or methanol (3.30 ppm) as an internalreference. The peak shapes and descriptors for the peak shapes aredenoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; c, complex; br, broad; app, apparent. Low-resolution massspectra were obtained under thermospray (TS) conditions on a Fisons (nowMicromass) Trio 1000 Mass Spectrometer (Micromass Inc., Beverly, Mass.),under chemical-ionization (CI) conditions on a Hewlett Packard 5989AParticle Beam Mass Spectrometer (Hewlett Packard Co., Palo Alto,Calif.), or under atmospheric pressure chemical ionization (APCI) on aFisons (now Micromass) Platform II Spectrometer. Optical rotations wereobtained on a Perkin-Elmer 241 MC Polarimeter (Perkin-Elmer, Norwalk,Conn.) using a standard path length of 1 dcm at about 23° C. at theindicated concentration in the indicated solvent.

Liquid column chromatography was performed using forced flow (flashchromatography) of the indicated solvent on either Baker Silica Gel (40μm, J. T. Baker, Phillipsburg, N.J.) or Silica Gel 60 (EM Sciences,Gibbstown, N.J.) in glass columns or using low nitrogen or air pressurein Flash 40™ or Flash 12™ (Biotage, Charlottesville, Va.) cartridges.Radial chromatography was performed using a Chromatron (HarrisonResearch, Palo Alto, Calif.). The terms “concentrated” and “evaporated”refer to removal of solvent using a rotary evaporator at water aspiratorpressure or at similar pressures generated by a Büchi B-171 Vacobox(Brinkmann Instruments, Inc., Westbury, N.Y.) or a Büchi B-177 Vacoboxwith a bath temperature equal to or less than 50° C. Reactions requiringthe use of hydrogen gas at pressures greater than 1 atmosphere were runusing a Parr hydrogen apparatus (Parr Instrument Co., Moline, Ill.).Unless otherwise specified, reagents were obtained from commercialsources. The abbreviations “d”, “h”, and “min” stand for “day(s)”,“hour(s)”, and “minute(s)”, respectively.

EXAMPLE 14-(4-Hydroxymethyl-6-[1,3,5]triazin-2-yl)-piperazine-1-sulfonic aciddimethylamide Step 1 4-Dimethylsulfamoyl-piperazine-1-carboxylic aciddimethylamide

To a solution of N-N-dimethylsulfamoyl-piperazine (5.2 mmol, 1.0 g) inTHF (10 mL) and triethylamine (0.75 mL) was addedN,N-dimethylaminocarbamoyl chloride (0.5 mL) and the reaction wasstirred for 2 h at room temperature. The precipitated triethylaminehydrochloride was filtered off and the filtrate was evaporated to obtaina white solid, which was crystallized from a 1:1 mixture of EtOAc andn-hexane to yield the title compound of Step 1 (88%).

Step 2 4-(Chloro-dimethylamino-methylene)-piperazine-1-sulfonic aciddimethylamide hydrochloride

A mixture of the title compound of Step 1 (2 mmol, 530 mg) andphosphorus oxychloride (2 mmol, 0.2 mL) was heated to 110° C. for 0.5 h.After cooling the reaction solidified to yield the title compound ofStep 2, which was immediately used in Step 4 below.

Step 3 2-Methoxy-N-cyano-acetamidine

To an ice-cold solution of 2-methoxyacetamidine hydrochloride (0.1 mol)in ethanol (100 mL) and triethyl amine (0.2 mol, 27.8 mL) was addeddropwise a solution of cyanogen bromide in acetonitrile. After 1 hr thesolvents and the excess triethylamine were removed by evaporation andwater (100 mL) was added to the resulting residue. It was then extractedwith EtOAc (2×100 mL). The EtOAc extract was collected, dried, filteredand the filtrate was evaporated to obtain a light yellow solid, thetitle product of Step 3 (72%, 8.2 g); mp 101-103° C.

Step 42-Chloro-4-(4-methoxymethyl-6-[1,3,5]triazin-2-yl)-piperazine-1-sulfonicacid dimethylamide

The solid title compound of Step 2 was dissolved in acetonitrile (10mL), to it was added all of the title compound of Step 3 and refluxedfor 2 h. After evaporating the excess acetonitrile, a residue wasobtained, which was purified by silica gel chromatography to yield thetitle compound of Step 4 (58%, 410 mg); mp, 143-144° C.

Step 5 4-(4-Methoxymethyl-6-[1,3,5]triazin-2-yl)-piperazine-1-sulfonicacid dimethylamide

A mixture of the title compound of Step 4 (1.0 mmol, 350 mg),palladium-carbon (100 mg), ethanol (10 mL), and sodium acetate (2.4mmol, 196 mg) was hydrogenated in a Parr shaker at 45 lbs./sq. inch(about 3.1 atm) for 1 h. The catalyst was filtered off and the filtratewas concentrated. The resulting white precipitate was filtered and theresidue was purified by silica gel chromatography (eluent,methanol/methylene chloride, 9:1) to obtain the title compound of Step 5(70%, 224 mg); mp, 78-81° C.; NMR 2.8 (s, 6H), 3.3 (m, 4H), 3.98 (s,3H), 3.9 (m, 4H), 4.4 (s, 2H), 8.55 (s, 1H).

Step 6 4-(4-Hydroxymethyl-6-[1,3,5]triazin-2-yl)-piperazine-1-sulfonicacid dimethylamide

To an ice-cold solution of the title compound of Step 5 (1.5 mmol, 474mg) in methylene chloride (20 mL) was added dropwise a solution of borontribromide (1M in methylene chloride, 3 mL). After 2 h, the reaction wasquenched with water (5 mL) and sufficient 10% potassium hydroxidesolution to raise the pH to 9. The methylene chloride layer wascollected, dried, filtered and the filtrate was evaporated to a solidresidue. This was crystallized from acetone to obtain the title compoundof Step 6 and this Example (68%, 275 mg); mp, 157-159° C.

EXAMPLE 21-{4-[3R,5S-Dimethyl-4-(4-methyl-[1,3,5]triazin-2-yl)-piperazin-1-yl]-[1,3,5]triazin-2-yl}-(R)ethanol

To an ice-cold solution of 2-methoxy-propinamidine (3.8 mmol, 523 mg) inabsolute ethanol (5 mL) and triethylamine (7.6 mmol, 1.1 mL) was addedcyanogen bromide (2.9 M in methylene chloride, 1.3 mL). After theaddition, the reaction temperature was slowly raised to room temperatureand stirred for 3 hr. Evaporation of all volatile liquids gave a solidresidue, which was extracted with EtOAc. The EtOAc layer was washed withwater and the EtOAc layer was collected, dried, filtered and thefiltrate was evaporated to dryness to obtain2-methoxy-N-cyanopropinamidine (85%, 410 mg).

To an ice-cold solution of 2,6-dimethyl piperazine (179 mmol, 20.4 g),methylene chloride (200 mL), and triethylamine (214 mmol, 29.9 mL) wasadded dropwise dimethylcarbamoyl chloride (179 mmol, 16.4 mL). After 4hr the reaction was quenched with a saturated sodium bicarbonatesolution and the methylene chloride layer was collected, dried, filteredand the filtrate was evaporated to dryness to obtain an orange oil,2,6-dimethyl-piperazine-1-carboxylic acid dimethylamide (70%, 23.1 g).

A mixture of 2,6-dimethyl-piperazine-1-carboxylic acid dimethylamide andphosphorus oxychloride (51 mmol, 4.8 mL) was heated at 110° C. for 30min. After cooling the reaction to room temperature,2-methoxy-N-cyanopropinamidine (51 mmol, 6.5 g) and acetonitrile wasadded and then refluxed for 2 hr. The reaction mixture was evaporated todryness and the residue was purified by silica gel chromatography(eluent, 9:1 methylene chloride-methanol) to obtain2-chloro-4-(3,5-dimethyl-piperazin-1-yl)-6-(1-methoxy-ethyl)-[1,3,5]triazine(24%, 5.1 g).

A mixture of2-chloro-4-(3,5-dimethyl-piperazin-1-yl)-6-(1-methoxy-ethyl)-[1,3,5]triazine(1.12 mmol, 321 mg), 2,4-dichloro-6-methyl triazine (1.12 mmol, 184 mg),sodium bicarbonate (2.24 mmol, 189 mg) and DMF (3 mL) was stirred atroom temperature overnight and was diluted with EtOAc (20 mL) and water(30 mL). The EtOAc extract was collected, dried, filtered and thefiltrate was evaporated to a residue, which was purified by silica gelchromatography (eluent, 99:1 methylene chloride-methanol) to obtain2-chloro-4-[2-(4-chloro-6-methyl-[1,3,5]triazine-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-6-(1-methoxy-ethyl)-[1,3,5]triazine(49%, 225 mg).

A mixture of2-chloro-4-[2-(4-chloro-6-methyl-[1,3,5]triazine-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-6-(1-methoxy-ethyl)-[1,3,5]triazine(051 mmol, 211 mg), Pd—C catalyst (10%, 84 mg), HCl (2 M in ether, 0.76mmol, 0.38 mL), ammonium formate (5.1 mmol, 322 mg) and isopropanol (8mL) was stirred at 90° C. for 2 h. After cooling the reaction it wasdiluted with methylene chloride (20 mL) and was filtered. The filtratewas evaporated to dryness and the residue was partitioned betweenchloroform and aq. saturated sodium bicarbonate. The chloroform layerwas collected, dried, filtered and the filtrate was evaporated to aresidue, which was purified by silica gel chromatography (eluent, 99:1methylene chloride-methanol) to obtain2-[3R,5S-dimethyl-2-(4-methyl-)-[1,3,5]triazine-4-yl)-piperazin-1-yl]-4-(1-methoxy-ethyl)-[1,3,5]triazine(97%, 170 mg).

2-[3R,5S-Dimethyl-2-(4-methyl-)-[1,3,5]triazine-4-yl)-piperazin-1-yl]-4-(1-methoxy-ethyl)-[1,3,5]triazinewas deprotected according to the procedures set forth in Step 6 ofExample 1 above to obtain1-[4-[3R,5S-dimethyl-4-(4-methyl-[1,3,5]triazine-2-yl)-piperazin-1-yl]-[1,3,5]triazin-2-yl}-(R,S)ethanol, which was chromatographed (HPLC) using a chiral column toobtain the title compound of this Example. (76%; mp, 136-138° C.;[α]_(D)+14.4 (1.19 mg/ml, methanol)).

EXAMPLE 31-{4-[4-(4-Cyclopropyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol

To an ice-cold solution of2-chloro-4-(3,5-dimethyl-piperazin-1-yl)-6-(1-methoxy-ethyl)-[1,3,5]triazine,prepared in Example 2 above, (2.2 mmol, 631 mg) in methylene chloride (7mL) was added boron tribromide (1 M in methylene chloride, 11.04 mmol,11 mL) and the reaction was stirred for 2 h. After the reaction wasallowed to come to room temperature, methylene chloride (10 mL) wasadded to it followed by a small quantity of water (1 mL) to quench theunreacted boron tribromide. Sufficient saturated aq. sodium bicarbonatewas added to raise the pH of the reaction solution to 8. The methylenechloride layer was collected, dried, filtered and the filtrate wasevaporated to dryness to obtain a residue, which was purified by silicagel chromatography (eluent, 98:2 methylene chloride-methanol) to obtain1-[4-chloro-6-(3,5-dimethyl-piperazin-1-yl)-[1,3,5]triazin-2-yl]-ethanol(65%, 392 mg); mp 126-128° C.

A mixture of this compound (0.74 mmol, 200 mg),2,4-dichloro-6-cyclopropyl-triazine (0.74 mmol, 140 mg), sodiumbicarbonate (1.47 mmol, 124 mg) and DMF (4 mL) was stirred at roomtemperature overnight and was diluted with EtOAc (20 mL) and water (20mL). The EtOAc extract was collected, dried, filtered and the filtratewas evaporated to a white solid,2-chloro-4-[2-(4-chloro-6-cyclopropyl-[1,3,5]triazine-2-yl)-3R,S5-dimethyl-piperazin-1-yl]-6-(1-methoxy-ethyl)-[1,3,5]triazine(98%, 306 mg).

A mixture of2-chloro-4-[2-(4-chloro-6-cyclopropyl-[1,3,5]triazine-2-yl)-3R,S5-dimethyl-piperazin-1-yl]-6-(1-methoxy-ethyl)-[1,3,5]triazine(0.72 mmol, 306 mg), Pd—C catalyst (10%, 122 mg), HCl (2 M in ether,1.08 mmol, 0.54 mL), ammonium formate (7.2 mmol, 454 mg) and isopropanol(7 mL) was stirred at 90° C. for 2 h. After cooling the reaction, it wasdiluted with methylene chloride (20 mL) and was filtered. The filtratewas evaporated to dryness and the residue was partitioned betweenchloroform and aq. saturated sodium bicarbonate. The chloroform layerwas collected, dried, filtered and the filtrate was evaporated to aresidue, which was purified by silica gel chromatography (eluent, 99:1methylene chloride-methanol) to obtain a solid, which was trituratedwith isopropyl ether to obtain the title compound of this Example (64%,106 mg); mp 120-121° C.

EXAMPLE 4Benzofuran-2-yl-{4-[4-1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-methanone

A mixture of1-[4-chloro-6-(3,5-dimethyl-piperazin-1-yl)-[1,3,5]triazin-2-yl]-ethanol,prepared in Example 3 above (1.26 mmol, 457 mg), benzofuran-2-carboxylicacid chloride (1.26 mmol, 228 mg), triethylamine (2.52 mmol, 0.35 mL)and methylene chloride (6 mL) was stirred overnight at room temperature.After adding a further quantity of methylene chloride (10 mL) and aq.saturated sodium bicarbonate to the reaction, the methylene chloridelayer was collected, dried, filtered and the filtrate was evaporated todryness to obtain a brown oil (650 mg), which was immediately used inthe next step. A mixture of this brown oil, Pd—C catalyst (10%, 1.3 g),HCl (2 M in ether, 2 mL), ammonium formate (1.6 g), and isopropanol (15mL) was heated at 90° C. for 1 h. After cooling the reaction, it wasdiluted with methylene chloride (20 mL) and was filtered. The filtratewas evaporated to dryness and the residue was partitioned betweenchloroform and aq. saturated sodium bicarbonate. The chloroform layerwas collected, dried, filtered and the filtrate was evaporated to aresidue, which was purified by silica gel chromatography (eluent, 99:1methylene chloride-methanol) to obtain a solid, which was trituratedwith isopropyl ether to obtain the title product of this Example (24%,84 mg); mp, 99-101° C.

EXAMPLE 4AFuro[2,3-c]pyridin-2-yl-{4-[4-1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-methanone

The title compound of this example was prepared according to proceduresanalogous to those described in Example 4, exceptfuro[2,3-c]pyridin-2-carboxylic acid chloride was used in place ofbenzofuran-2-carboxylic acid chloride, mp, 99-101° C.

EXAMPLE 54-[4-(1-Hydroxy-ethyl)-[1,3,5]triazin-2-yl]-piperazine-1-sulfonic aciddimethylamide

To a suspension of 2-benzyloxy-propionamide (Helv. Chim. Acta, 1971,845-851) (26.6 mmol, 4.77 g) in acetonitrile (100 mL) at roomtemperature was added dropwise chlorosulfonyl isocyanate (26.6 mmol, 4.1mL) in acetonitrile (20 mL). After 1 h the reaction was concentrated,then carefully quenched with water (20 mL) and allowed to stir at roomtemperature for 1 h. The precipitated solid was filtered, collected andair-dried to obtain (2-benzyloxy-propionyl)-urea (62%, 3.66 g); NMR 1.2(d, 3H), 4.0 (t, 1H), 4.4 (dd, 2H), 7.3 (m, 5H), 7.7 (s, 1H), 10.0 (S,1H). The above reaction conditions were followed to convert(2-benzyloxy-propionyl)-urea to2-benzyloxy-N-ureidocarbonyl-propionamide, using the above compound,(2-benzyloxy-propionyl)-urea (16.5 mmol, 3.66 g), chlorosulfonylisocyanate (28.8 mmol, 2.5 mL), and acetonitrile (80 mL). The yield of2-benzyloxy-N-ureidocarbonyl-propionamide was 59% (2.56 g); NMR 1.4 (d,3H), 4.2 (t, 1H), 4.6 (dd, 2H), 7.4 (m, 5H), 9.8 (s, 1H), 11.1 (s, 1H).

To an ice-cold suspension of 2-benzyloxy-N-ureidocarbonyl-propionamide(9.4 mmol, 2.5 g) in water (15 mL) was added KOH (28 mmol, 1.6 g) inwater (10 mL). The reaction temperature was slowly raised to roomtemperature, and the reaction was allowed to stir for 1 h. Sufficientacetic acid was added to adjust the pH of the reaction to 5, and theresulting cloudy solution was extracted with chloroform (3×20 mL). Thechloroform layer was collected, dried, filtered and the filtrate wasconcentrated to obtain a residue, which was triturated with isopropylether to obtain 6-(1-benzyloxy-ethyl)-1H-[1,3,5]triazine-2,4-dione (74%,1.72 g); NMR 1.4 (d, 3H), 4.2 (t, 1H), 4.6 (dd, 2H), 7.4 (m, 5H), 11.2(s, 1H), 12.1 (s, 1H).

A mixture of 6-(1-benzyloxy-ethyl)-1H-[1,3,5]triazine-2,4-dione (6.1mmol, 1.5 g), phosphorus oxychloride (18.2 mmol, 1.7 mL), and diethylaniline (1 mL) was heated at 70° C. for 1 h. Excess phosphorusoxychloride was removed and the residual oil was extracted withchloroform (2×20 mL); the extract was washed with water (3×20 mL); thechloroform layer was collected, dried, filtered and the filtrate wasevaporated to obtain an oily product. This oily product waschromatographed over silica gel (eluent, 9:1 hexane-EtOAc) to obtain2-(1-benzyloxy-ethyl)-4,6-dichloro-[1,3,5]triazine (35%, 611 mg); NMR1.6 (d, 3H), 4.0 (t, 1H), 4.6 (dd, 2H), 7.3 (m, 5H).

A mixture of 2-(1-benzyloxy-ethyl)-4,6-dichloro-[1,3,5]triazine (0.75mmol, 212 mg), NN-dimethylsulfamoyl piperazine (0.75 mmol, 144 mg),sodium bicarbonate (1.5 mmol, 125 mg), and DMF (3 mL) was stirredovernight at room temperature. EtOAc (15 mL) and water (20 mL) wereadded and the EtOAc extract was collected and washed with water (2×10mL). The EtOAc layer was collected, dried and filtered, and the filtratewas evaporated to obtain4-[4-(1-benzyloxy-ethyl)-6-chloro-[1,3,5]triazin-2-yl]-piperazine-1-sulfonicacid dimethylamide (97%, 320 mg); mass spectrum, m/e 441.

A mixture of4-[4-(1-benzyloxy-ethyl)-6-chloro-[1,3,5]triazin-2-yl]-piperazine-1-sulfonicacid dimethylamide (0.73 mmol, 320 mg), (Pd—C 910%, 640 mg), HCl (2 M inether, 4.4 mmol, 2.2 mL), ammonium formate (15 mmol, 915 mg) andisopropanol (10 mL) was heated at 90° C. for 2 h. The reaction wascooled and filtered, and to the filtrate was added chloroform (20 mL)and aq. saturated sodium bicarbonate (20 mL). The chloroform layer wascollected, dried, filtered and the filtrate was evaporated to dryness.The resulting residue was purified by silica gel chromatography (eluent,96:4 chloroform-methanol) to yield the title compound of this example(59%, 136 mg); mp 124-125° C.; NMR 1.5 (d, 3H), 2.8 (s, 6H), 3.3 (m,4H), 4.0 (s, 4H0, 7.2 (s, 1H, 8.6 (s, 1H).

EXAMPLE 61-{4-[4-(4-Hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol

A mixture of1-[4-chloro-6-(3,5-dimethyl-piperazin-1-yl)-[1,3,5]triazin-2-yl]-ethanol,prepared in Example 3 (0.78 mmol, 212 mg),2,4-dichloro-6-diazomethyl-triazine (0.78 mmol, 148 mg), sodiumbicarbonate (1.56 mmol, 131 mg), and DMF (3 mL) was stirred overnight atroom temperature. EtOAc (15 mL) and water (20 mL) were added and theEtOAc extract was collected and washed with water (2×10 mL). The EtOAclayer was collected, dried and filtered, and the filtrate was evaporatedto obtain an oily product,2-{4-[4-(4-chloro-6-diazomethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethylpiperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol(442 mg); mass spectrum m/e 425.

The crude oily product,2-{4-[4-(4-chloro-6-diazomethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethylpiperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol,was dissolved in EtOAc (10 mL) and to the solution was added 10%sulfuric acid (2 mL) and allowed to stir for 1 h. Excess EtOAc wasremoved and the residue was partitioned between methylene chloride (20mL) and aq. saturated sodium bicarbonate (10 mL). The methylene chloridelayer was collected, dried and filtered, and the filtrate was evaporatedto obtain an oily product,2-{4-[4-(4-chloro-6-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethylpiperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol(43%, 184 mg), which was used in the next step without furtherpurification, mass spectrum m/e 415.

The above product,2-{4-[4-(4-chloro-6-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethylpiperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol,was dechlorinated according to the procedures described in Example 3above to obtain the title compound of this example (18%, 28 mg); mp188-192° C.

EXAMPLE 72-{4-[4-(4-Hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-6-methyl-[1,3,5]triazin-2-yl}-phenol

A mixture of 2-methoxy-benzonitrile (75.6 mmol, 10.06 g),trichloroacetonitrile (151.1 mmol, 21.8 g), and aluminum tribromide(0.76 mmol, 201 mg) was cooled to −20° C. and HCl gas was bubbled intothe mixture for 20 min. After stirring the reaction mixture for 2 h at−20° C., the temperature of the reaction was allowed to come to roomtemperature. After overnight stirring, the reaction was quenched withwater (100 mL) and extracted with EtOAc (2×200 mL). The EtOAc extractwas washed with aq. saturated sodium bicarbonate (2×20 mL) and the EtOAclayer was collected, dried and filtered, and the filtrate was evaporatedto a solid residue, which was purified by silica gel chromatography(eluent, 4:1 hexane-EtOAc) to obtain1-(2-methoxyphenyl)-3,5-bis-trichloromethyl-triazine (26%, 8.4 g); mp93-95° C.

A mixture of 1-(2-methoxyphenyl)-3,5-bis-trichloromethyl-triazine (4.74mmol, 2 g), 2,6-dimethyl piperazine (4.74 mmol, 541 mg), sodiumbicarbonate (9.48 mmol, 797 mg), and DMF (10 mL) was stirred overnightat room temperature. Water (30 mL) and EtOAc (30 mL) were added to thereaction. The EtOAc layer was collected, dried and filtered, and thefiltrate was evaporated to obtain a thick liquid, which was2-(3,5-dimethyl-piperazin-1-yl)-4-(2-methoxy-phenyl)-6-trichloromethyl-[1,3,5]triazine(95%, 1.88 g); mass spectrum m/e 416.

The above thick liquid (4.51 mmol, 1.88 g), Pd—C catalyst (10%, 752 mg),HCl (2 M in ether, 3.4 mL), ammonium formate (2.84 g), and methanol (50mL) were refluxed for 1 h. After cooling the reaction, it was filtered.The filtrate was evaporated to dryness and the residue was partitionedbetween chloroform (100 mL) and aq. saturated sodium bicarbonate. Thechloroform layer was collected, dried and filtered, and the filtrate wasevaporated to obtain a solid, which was2-(3,5-dimethyl-piperazin-1-yl)-4-(2-methoxy-phenyl)-6-methyl-[1,3,5]triazine(82%, 1.16 g); mass spectrum m/e 313.

To an ice-cold solution of2-(3,5-dimethyl-piperazin-1-yl)-4-(2-methoxy-phenyl)-6-methyl-[1,3,5]triazine(3.57 mmol, 1.12 g) in methylene chloride (50 mL) was added dropwiseboron tribromide (1 M in methylene chloride, 17.9 mL) and stirred for 2h. The reaction was diluted with methylene chloride (100 mL), quenchedwith water (30 mL) and saturated bicarbonate solution (20 mL). Themethylene chloride layer was collected, dried, filtered, and thefiltrate was evaporated to obtain a brown solid, which was purified bysilica gel chromatography (eluent, 98:2 chloroform-methanol) to obtain atan solid, which was2-[4-(3,5-dimethyl-piperazin-1-yl)-6-methyl-[1,3,5]triazin-2-yl]-phenol(37%, 391 mg); mass spectrum m/e 299.

A mixture of2-[4-(3,5-dimethyl-piperazin-1-yl)-6-methyl-[1,3,5]triazin-2-yl]-phenol(0.84 mmol, 250 mg), 2,4-dichloro-6-diazomethyl-triazine (0.84 mmol, 159mg), sodium bicarbonate (1.67 mmol, 317 mg), and DMF (5 mL) was stirredovernight at room temperature. EtOAc (15 mL) and water (20 mL) wereadded and the EtOAc extract was collected and washed with water (2×10mL). The EtOAc layer was collected, dried and filtered, and the filtratewas evaporated to obtain a brown semi-solid (183 mg), which was2-{4-[4-(4-chloro-6-diazomethyl-[1,3,5]triazin-2-yl)-3,5-dimethyl-piperazin-1-yl]-6-methyl-[1,3,5]triazin-2-yl}-phenol,mass spectrum m/e 452.

The brown semi-solid (183 mg) was dissolved in EtOAc (10 mL) and to thesolution was added 10% sulfuric acid (2 mL) and allowed to stir for 1hr. Excess EtOAc was removed and the residue was partitioned betweenmethylene chloride (20 mL) and aq. saturated sodium bicarbonate (10 mL).The methylene chloride layer was collected, dried and filtered, and thefiltrate evaporated to obtain an oily product (36%, 64 mg), which was2-{4-[4-(4-chloro-6-hydroxymethyl-[1,3,5]triazin-2-yl)-3,5-dimethyl-piperazin-1-yl]-6-methyl-[1,3,5]triazin-2-yl}-phenol,and which was used in the next step without further purification; massspectrum m/e 443.

A mixture of2-{4-[4-(4-chloro-6-hydroxymethyl-[1,3,5]triazin-2-yl)-3,5-dimethyl-piperazin-1-yl]-6-methyl-[1,3,5]triazin-2-yl}-phenol(0.145 mmol, 64 mg), Pd—C catalyst (10%, 64 mg), HCl (2 M in ether,0.217 mmol, 0.11 mL), ammonium formate (1.45 mmol, 91 mg) andisopropanol (5 mL) was stirred at 90° C. for 2 h. After cooling thereaction, it was diluted with methylene chloride (20 mL) and wasfiltered. The filtrate was evaporated to dryness and the residue waspartitioned between chloroform and aq. saturated sodium bicarbonate. Thechloroform layer was collected, dried and filtered, and the filtrate wasevaporated to a residue, which was purified by silica gel chromatography(eluent, 99:1 methylene chloride-methanol) to obtain a solid, which wastriturated with isopropyl ether to obtain the title compound of thisexample (54%, 32 mg); mp 188-190° C.

EXAMPLE 8 Dimethylamino-acetic acid1-{4-[3R,5S-dimethyl-4-(4-methyl-[1,3,5]triazin-2-yl)-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethylester

A mixture of the title compound of Example 2,1-{4-[3,5-dimethyl-4-(4-methyl-[1,3,5]triazin-2-yl)-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol,(0.45 mmol, 150 mg), N,N-dimethylaminoacetyl chloride (2.7 mmol, 440 mg)and triethyl amine (5.4 mmol, 0.75 mL) was refluxed overnight. Thereaction mixture was evaporated to a residue, which was purified bysilica gel chromatography (eluent, 94:6 methylene chloride-methanol) toobtain the title compound of this example (which is also the prodrug ofthe title compound of Example 2) as a viscous oil (30%, 56 mg); NMR 1.2(m, 6H), 1.6 (d, 3H0, 2.4 (m, 9H), 3.3 (s, 2H), 4.7 (m, 2H), 5.0 (m,2H), 5.6 (q, 1H), 8.45 (s, 1H), 8.55(s, 1H).

EXAMPLE 91-(4-{4-[4-(1-Hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-[1,3,5]triazin-2-yl)-ethanol

A mixture of2-chloro-4-(3,5-dimethyl-piperazin-1-yl)-6-(1-methoxy-ethyl)-[1,3,5]triazine,prepared as described in Example 2 above (1.45 mmol, 525 mg),2-(1-benzyloxy-ethyl)-4,6-dichloro-[1,3,5]triazine, prepared asdescribed in Example 5 above (1.45 mmol, 412 mg), sodium bicarbonate(291 mmol, 243 mg), and DMF (8 mL) was stirred overnight at roomtemperature. EtOAC (30 mL) and water (30 mL) were added to the reactionmixture. The EtOAc layer was collected, dried, filtered and evaporatedto dryness to obtain a yellow oil,1-{4-(4-[4-(1-benzyloxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl-3-(1-methoxy-ethyl)}-[1,3,5]triazine(92%, 812 mg); mass spectrum m/e 609.

A mixture of this oil,1-{4-(4-[4-(1-benzyloxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl-3-(1-methoxy-ethyl)}-[1,3,5]triazine(0.279 mmol, 170 mg), Pd—C catalyst (10%, 200 mg), HCl (2 M in ether,0.837 mmol, 0.42 mL), ammonium formate (5.58 mmol, 352 mg) andisopropanol (6 mL) was stirred at 90° C. for 2 h. After cooling thereaction, it was diluted with methylene chloride (20 mL) and wasfiltered. The filtrate was evaporated to dryness and the residue waspartitioned between chloroform and aq. saturated sodium bicarbonate. Thechloroform layer was collected, dried and filtered, and the filtrate wasevaporated to a residue, which was purified by silica gel chromatography(eluent, 96:4 methylene chloride-methanol) to obtain a solid, which wastriturated with isopropyl ether to obtain the title compound of thisExample (44%, 44 mg); mp 142-144° C.

EXAMPLES 10-15

Following procedures analogous to those described above, particularly inExamples 2, 3, 6 and 9, the following compounds of the present inventionwere prepared:

Example No. R¹ R^(z1) R^(z2) Physico-chemical Data 10 H H Ph mp 206-207°C. 11 H H CH₂OH mp 224-225° C. 12 H CH₃ OCH₃ mp 155-160° C. 13 CH₃ H PhNMR 1.14(s, 3H), 1.22(s, 3H), 1.28(d, 3H), 3.24(m, 2H), 3.9(s, 1H), 4.659q, 1H), 4.8(m, 2H), 5.1(s, 2H), 7.5(m, 3H), 8.4(m, 2H0, 8.55(s, 1H),8.68(s, 1H) 14 CH₃ OH Ph mp >275° C. 15 CH₃ OH CH₃ mp 263-265° C. Theabove compounds in Examples 10-15 may be named as follows: Example 101-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;Example 111-{4-[4-(4-Hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;Example 121-{4-[4-(4-methoxy-6-methyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;Example 131-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;Example 141-{4-[4-(4-Hydroxy-6-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;Example 151-{4-[4-(4-Hydroxy-3-methyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol.

EXAMPLES 16 AND 17

Following procedures analogous to those described above, particularly inExample 7, the following compounds of the invention were prepared:

Example No. R¹ R^(z1) R^(z2) Physico-chemical Data 16 H H Ph mp,197-198° C. 17 H H CH₂OMe mp, 145-150° C. The above compounds inExamples 16 and 17 may be named as follows: Example 161-{4-[4-(4-Phenyl-[1,3,5]triazin-2-yl)-2R,6S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;Example 171-{4-[4-(4-methoxymethyl-[1,3,5]triazin-2-yl)-2R,6S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol.

1. A compound of formula I

a stereoisomer thereof, a prodrug of said compound or stereoisomer, or apharmaceutically acceptable salt of said compound, stereoisomer orprodrug; wherein R¹ is a) hydrogen or b) —C₁-C₄)alkyl; R² and R³ areeach independently a) hydrogen, b) —(C₁-C₄)alkyl, c) —(C₃-C₆)cyoloalkylor d) phenyl which for each occurrence is optionally substituted withone or two substituants, each substituent is independently selected fromGroup Q; X is a) —C(O)—R⁴-Z, b) —SO₂—R⁴-Z, c) —C(O)—NR⁵R⁶, d) —SO₂—NR⁵R⁶or e) 1,3,5-triazin-2-yl having R^(z1) and R^(z2) substituents; R⁴ is a)a covalent bond or b) —(C₁-C₄)alkyl-; Z is a) phenyl or benzyl whereinthe phenyl ring in each of these groups is optionally substituted withone or two substitutents, each substituent is independently selectedfrom Group Q, or b) Het; R⁵ and R⁶ are each independently a) hydrogen,b) —(C₁-C₄)alkyl or c) (C₃-C₈)cycloalkyl; or R⁵ and R⁶ are takentogether along with the nitrogen atom to which they are attached to formpyrrolidinyl or piperidinyl; Het is a) pyridyl, b) thiazolyl, c)oxazolyl, d) quinolyl, e) isoqulnolyl, f) phthalizinyl, g) quinoxalyl,h) benzthlazolyl, i) benzoxazolyl, j) benzofuranyl, k) benzothinyl, l),furanopyridyl or m) thienopyridyl; wherein each of these groups isoptionally substituted with one or two substituents, each substituent isindependently selected from Group Q; Group Q is a) fluoro, b) chloro, c)bromo, d) —(C₁-C₄)alkyl, e) —(C₃-G₆)cycloalkyl, f) —O—(C₁-C₄)alkyl, g)—S—(C₁-C₄)alkyl, h) —SO₂—(C₁-C₄)alkyl, i) hydroxy or j)—(C₁-C₄)alkyl-hydroxy; R^(z1) and R^(z2) are each independently selectedfrom a) hydrogen, b) hydroxy, c) chloro, d) —(C₁-C₄)alkyl, e)—(C₃-C₆)cycloalkyl, f) —O—(C₁-C₄)alkyl, g) —(C₁-C₄)alkyl-O—(C₁-C₄)alkyl,h) —CHO, i) —C(O)—(C₁-C₄)alkyl, j) —(C₁-C₆)alkyl-hydroxy, k) phenylwhich for each occurrence is optionally substituted with one or twosubstitutents, each substituent is independently selected from Group Q,l) pyrroyl, m) imidazolyl or n) triazolyl.
 2. A compound of claim 1wherein R¹ is hydrogen or methyl.
 3. A compound of claim 2 wherein R²and R³ are each independently a) hydrogen, b) —(C₁-C₄)alkyl, c)—(C₃-C₆)cycloalkyl; or d) phenyl optionally substituted with one or twosubstituents, each substituent is independently selected from 1)—(C₁-C₄)alkyl, 2) —(C₃-C₆)cycloalkyl, 3) —O—(C₁-C₄)alkyl, 4) fluoro or5) chloro.
 4. A compound of claim 3 wherein R² and R³ are eachindependently hydrogen or methyl.
 5. A compound of claim 4 wherein R² ishydrogen and R³ is hydrogen.
 6. A compound of claim 4 wherein R² ismethyl and R³ is methyl.
 7. A compound of claim 4 wherein X is1,3,5-triazin-2-yl having R^(z1) and R^(z2) substituents.
 8. A compoundof claim 7 wherein one of the R^(z1) and R^(z2) substituents is hydrogenand the other is methyl, cyclopropyl, —CH₂OH, —CH(CH₃)OH or phenyl.
 9. Acompound of claim 7 wherein one of the R^(z1) and R^(z2) substituents ismethyl and the other is methoxy or phenyl optionally substituted with2-hydroxy.
 10. A compound of claim 7 wherein one of the R^(z1) andR^(z2) substituents is hydroxy and the other is methyl or phenyl.
 11. Acompound of claim 4 wherein X is —SO₂N(CH₃)₂.
 12. A compound of claim 4wherein X is —C(═O)-benzofuranyl.
 13. A compound of claim 4 wherein X is—C(═O)-furanopyridyl.
 14. A compound selected from:4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-piperazine-1-sulfonic aciddimethylamide;1-{4-[3R,5S-dimethyl-4-(4-methyl-[1,3,5]triazin-2-yl)-piperazine-1-yl]-1,3,5]triazin-2-yl}-R-ethanol;1-{4-[4-(4-cyclopropyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;benzofuran-2-yl-{4-[4-1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-methanone;4-[4-(1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-piperazine-1-sulfonic aciddimethylamide;1-{4-[4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;2-{4-[4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-6-methyl-[1,3,5]triazin-2-yl}-phenol;dimethylamino-acetic acid1-{4-[3R,5S-dimethyl-4-(4-methyl-[1,3,5]triazin-2-yl)-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethylester;1-(4-{4-[4-(1-hydroxy-ethyl)-[1,3,5,]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-[1,3,5]triazin-2-yl)-ethanol;1-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-2R,6S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;1-{4-[4-(4-hydroxy-3-methyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;1-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;1-{4-[4-(4-methoxymethy-[1,3,5]triazin-2-yl)-2R,6S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;1-{4-[4-(4-hydroxymethyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;1-{4-[4-(4-methoxy-6-methyl-[1,3,5]triazin-2yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-methanol;1-{4-[4-(4-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;1-{4-[4-(4-hydroxy-6-phenyl-[1,3,5]triazin-2-yl)-3R,5S-dimethyl-piperazin-1-yl]-[1,3,5]triazin-2-yl}-ethanol;andfuro[2,3c]pyridin-2-yl-{4-[4-1-hydroxy-ethyl)-[1,3,5]triazin-2-yl]-2R,6S-dimethyl-piperazin-1-yl}-methanone.15. A pharmaceutical composition comprising a compound of claim 1, aprodrug thereof or a pharmaceutically acceptable salt of said compoundor said prodrug and a pharmaceutically acceptable vehicle, carrier ordiluent.
 16. A method of inhibiting sorbitol dehydrogenase in a mammalin need of such inhibition comprising administering to said mammal asorbitol dehydrogenase inhibiting amount of a compound of claim 1, aprodrug thereof or a pharmaceutically acceptable salt of said compoundor said prodrug.
 17. A method of treating diabetes in a mammal sufferingfrom diabetes comprising administering to said mammal an effectiveamount of a compound of claim 1, a prodrug thereof or a pharmaceuticallyacceptable salt of said compound or said prodrug.
 18. A method ofreducing tissue damage resulting from ischemia comprising administeringto a mammal in need of said treatment an effective amount of a compoundof formula I of claim 1, a prodrug thereof or a pharmaceuticallyacceptable salt of said compound or said prodrug; wherein said ischemiais a result of an etiology independent of diabetic microangiopathy ordiabetic macroanglopathy.
 19. A method of claim 18 wherein the tissue isheart, brain, liver, kidney, lung, gut, skeletal muscle, spleen,pancreas, retina or intestinal tissue.
 20. A method of providing acardioprotective effect in a mammal which comprises administering to themammal en effective amount of a compound of formula I of claim 1, aprodrug thereof or a pharmaceutically acceptable salt of said compoundor said prodrug.